Novel hemopoietin receptor protein, nr12

ABSTRACT

A novel hemopoietin receptor gene (NR12) was successfully isolated by extracting motifs conserved among the amino acid sequences of known hemopoietin receptors and by using the predicted sequence. The NR12 gene encodes two forms of proteins, a transmembrane type and a soluble type. The expression of the NR12 gene was detected in tissues containing hematopoietic cells. NR12 is a novel hemopoietin receptor molecule involved in the regulation of immune system and hematopoiesis in vivo. Thus, NR12 is useful in the search for novel hematopoietic factors that functionally bind to the NR12 receptor, and in the development of therapeutic drugs for diseases associated with immunity or hematopoiesis.

This application is a divisional of U.S. application Ser. Nos.12/205,799 and 12/205,753, both filed Sep. 5, 2008, each being adivisional of U.S. application Ser. No. 11/595,320, filed Nov. 9, 2006,which is a continuation of U.S. application Ser. No. 11/274,375, filedNov. 14, 2005, now abandoned, which is a divisional of U.S. applicationSer. No. 10/105,930, filed Mar. 25, 2002, now U.S. Pat. No. 7,045,595,which is a continuation-in-part of International Patent Application No.PCT/JP00/06654, filed Sep. 27, 2000, which claims the benefit ofJapanese Patent Application Nos. 11-273358, filed Sep. 27, 1999, and2000-240397, filed Aug. 3, 2000. Each of the prior U.S. applications isherein incorporated by reference.

TECHNICAL FIELD

The present invention relates to novel hemopoietin receptor proteins,and genes encoding them, as well as methods for producing and using thesame.

BACKGROUND

A large number of cytokines are known as humoral factors that regulateproliferation/differentiation of various cells, or that regulate themaintenance, activation, and death of differentiated mature cells. Thereare specific receptors for these cytokines, which are categorized intoseveral families based on their structural similarities (Hilton D. J.,in “Guidebook to Cytokines and Their Receptors” edited by Nicola N. A.(A Sambrook & Tooze Publication at Oxford University Press), 1994, p8-16).

On the other hand, as compared to the similarities of their receptors,the homology of the primary-structure among cytokines is quite low. Nosignificant amino acid homology has be observed, even among cytokinemembers that belong to the same receptor family. This explains thefunctional specificity of respective cytokines, as well as similaritiesamong cellular reactions induced by each cytokine.

Representative examples of the above-mentioned receptor families are thetyrosine kinase receptor family, hemopoietin receptor family, tumornecrosis factor (TNF) receptor family, and transforming growth factor(TGF) receptor family. Different signal transduction pathways have beenreported to be involved with each of these families. Among thesereceptor families, many receptors of the hemopoietin receptor family inparticular are expressed in blood cells and immunocytes, and theirligands, cytokines, are often termed as hemopoietic factors orinterleukins. Some of these hemopoietic factors or interleukins existwithin blood and are thought to be involved in systemic humoralregulation of hemopoietic or immune functions.

This contrasts with the belief that cytokines belonging to otherfamilies are often involved in only topical regulation. Some of thesehemopoietins can be taken as hormone-like factors, and representativepeptide hormones, such as the growth hormone, prolactin, or leptinreceptors, also belong to the hemopoietin receptor family. Because ofthese hormone-like systemic regulatory features, it is anticipated thatadministration of these hemopoietins can be applied to the treatment ofvarious diseases. Among the large number of cytokines known, those thatare presently being clinically applied include erythropoietin, G-CSF,GM-CSF, and IL-2. Combined with IL-11, LIF, and IL-12 that are currentlyunder consideration for clinical trials, and the above-mentioned peptidehormones, such as the growth hormone and prolactin, it can be envisagedthat by searching novel cytokines that bind to hemopoietin receptorsamong the above-mentioned various receptor superfamilies, it is possibleto find a cytokine that can be clinically applied with a higherefficiency.

As mentioned above, cytokine receptors have structural similaritiesamong the family members. Using these similarities, many investigationsare aimed at finding novel receptors. In particular, many receptors ofthe tyrosine kinase receptor family have already been cloned, using itshighly conserved sequence at the catalytic site (Matthews et al., Cell65(7):143-52, 1991). In comparison, hemopoietin receptors do not have atyrosine kinase-like enzyme activity domain in their cytoplasmicregions, and their signal transductions are known to be mediated throughassociations with other tyrosine kinase proteins existing freely in thecytoplasm. Though the sites on receptors binding with these cytoplasmictyrosine kinases, called JAK kinases group, are conserved among familymembers, the homology is not very high (Murakami et al., Proc. Natl.Acad. Sci. USA 88:11349-11353, 1991). Actually, the sequence that bestcharacterizes these hemopoietin receptors exists in the extracellularregion. In particular, a five amino acid motif, Trp-Ser-Xaa-Trp-Ser(wherein “Xaa” is an arbitrary amino acid; SEQ ID NO:21), is conservedin almost all of the hemopoietin receptors. Therefore, novel receptorsmay be obtained by searching for novel family members using this motifsequence. In fact, these approaches have already led to theidentification of the IL-11 receptor (Robb et al., J. Biol. Chem.271(23):13754-13761, 1996), the leptin receptor (Gainsford et al., Proc.Natl. Acad. Sci. USA 93(25):14564-8, 1996), and the IL-13 receptor(Hilton et al., Proc. Natl. Acad. Sci. USA 93(1):497-501, 1996).

SUMMARY

The present invention provides novel hemopoietin receptor proteins, andDNA encoding these proteins. The present invention also provides avector into which the DNA has been inserted, a transformant harboringthe DNA, and a method for producing recombinant proteins using thetransformant. The present invention also provides methods of screeningfor compounds that bind to the protein.

Initially, the inventors attempted to find a novel receptor usingoligonucleotides encoding the Trp-Ser-Xaa-Trp-Ser motif (WS motif; SEQID NO:21) as the probe by the plaque hybridization method, RT-PCRmethod, and so on. However, it was extremely difficult to strictlyselect only those to which all 15 nucleotides that encode the motifwould completely hybridize under the usual hybridization conditions,because the oligonucleotide “tggag(t/c)nnntggag(t/c)” (wherein “n” is anarbitrary nucleotide; SEQ ID NO:22) encoding the motif was short, havingjust 15 base pairs. Further, because the g/c content of theoligonucleotide was high, higher than usual annealing temperatureconditions were required to strictly select those sequences in which allthe 15 nucleotides hybridized completely to the oligonucleotide.Therefore, performing screening under normal hybridization experimentconditions was extremely difficult.

To solve these problems, the inventors searched for additional motifs,other than the site of the above-mentioned WS motif that is conserved inthe hemopoietin receptor family. The inventors found that a residue,either tyrosine or histidine, located 13 to 27 amino acids upstream ofthe WS motif in the extracellular region was highly conserved in thereceptor family. Furthermore, additional search for consensus sequencesthat are frequently found in the 6 amino acids from the above Tyr/H isresidue toward the C-terminus led to the identification of the followingconsensus sequence:(Tyr/His)-Xaa-(Hydrophobic/Ala)-(Gln/Arg)-Hydrophobic-Arg (hereinafter,abbreviated as the YR motif). However, this YR motif is not exactly aperfect consensus sequence, and the combination of the nucleotidesequences that encode the motif is very complicated. Therefore, it ispractically impossible to synthesize and provide oligonucleotides thatencode all of the amino acid sequences as probes for hybridization,which is a practical method for screening, or as primers aimed forRT-PCR.

Accordingly, the inventors looked for other approaches to practicallysearch for novel members of the hemopoietin receptor family using theabove two motifs as probes, and determined that it would be appropriateto perform a database search on the computer using partial amino acidsequences of known hemopoietin receptors, including both motifs as thequery. The inventors repeated TblastN searches on the gss and htgsdatabase in GenBank, using partial amino acid sequences from multipleknown hemopoietin receptors as the query. As a result, many positiveclones, including known hemopoietin receptors, were obtained in allcases. Next, the nucleotide sequence around those sequences which seemedto be positive at a high rate was converted to the amino acid sequence.Genes considered to encode members of the receptor family were selectedby BlastX search, in which the amino acid sequences (converted from thenucleotide sequences of the clones) were compared to those of knownhemopoietin receptors. According to the two-step Blast search above,human genome sequences encoding two clones of known hemopoietin receptorgenes and one clone of novel hemopoietin receptor gene were identified.Subsequently, specific oligonucleotide primers were designed based onthe exon sequences predicted from the obtained nucleotide sequence.Clones corresponding to the N-terminal region and C-terminal region ofNR12 were obtained by conducting 5′-RACE and 3′-RACE methods using theprimers, and cDNA libraries of human fetal liver, adult thymus, andadult testis as the templates. The complete nucleotide sequence of thefull-length cDNA was revealed by determining the nucleotide sequences ofboth clones, and connecting the sequence at the duplicated centerregion.

From structural analyses, at least three kinds of transcription productsderived from splice variants were recognized. A cDNA clone of thesesplice variants comprising 337 amino acids and potentially encoding asecretory form soluble receptor protein was named NR12.1; the other twoclones, comprising 428 amino acids and 629 amino acids respectively andeach encoding transmembrane form receptor proteins, were named NR12.2and NR12.3. Because repeated structure of cysteine residues, YR motif,WS motif, and so on, that are conserved in the extracellular region ofother family members were well conserved in the primary structure of allthe isolated cDNA clones of NR12, it was considered that these clonesencode typical hemopoietin receptors.

Subsequently, RT-PCR was performed using primer sets specific to NR12.1,NR12.2, and NR12.3, respectively, against mRNA derived from varioushuman tissue. Then, tissues expressing these genes were searched, andthe distribution and the expression pattern of the genes in each humantissue were analyzed. Finally, in order to discard the possibility ofnon-specific amplification and to quantify the amount of the RT-PCRproducts, the products of RT-PCR were subjected to Southern blottingusing cDNA fragments specific to the respective clones. The resultindicated that these clones are mainly expressed in hematopoietic cellline tissue and immune cell line tissue.

Furthermore, the present inventors succeeded in obtaining two clones(NR12.4 and NR12.5) encoding complete proteins that were 3 amino acidsdifferent from NR12.2 and NR12.3, respectively, by conducting PCRcloning against the cDNA library of human thymus (wherein five clonesgenerically named “NR12” were isolated).

Based on the above features of NR12, NR12 is presumed to be a novelhemopoietin receptor molecule related to the regulation of the immunesystem or hematopoiesis. The gene encoding NR12 will be extremely usefulin the screening for novel hematopoietic factors that can functionallybind to the receptor.

Moreover, the present inventors succeeded in isolating genomic fragmentsof mouse receptor homologues by conducting xenogenic cross hybridizationcloning using cDNA of human NR12 as the probe. It is expected thatfurther elucidation of the in vivo function of the receptor protein ispossible by constructing mutant mouse lacking NR12 gene using the mousegene fragments.

Consequently, the present invention relates to novel hemopoietinreceptors and genes encoding the receptors, as well as use of the same.More specifically, the present invention provides the following:

(1) a DNA selected from the group consisting of:

(a) a DNA encoding a protein comprising the amino acid sequence of anyone of SEQ ID NOs:2, 4, 6, 8, and 10;

(b) a DNA comprising the coding region of the nucleotide sequence of anyone of SEQ ID NOs:1, 3, 5, 7, and 9;

(c) a DNA encoding a protein comprising the amino acid sequence of anyone of SEQ ID NOs:2, 4, 6, 8, and 10, in which one or more amino acidsare modified by substitution, deletion, insertion, and/or addition,wherein said protein is functionally equivalent to the proteinconsisting of the amino acid sequence of any of SEQ ID NOs:2, 4, 6, 8,and 10; and,

(d) a DNA hybridizing under stringent conditions with a DNA consistingof the nucleotide sequence of any one of SEQ ID NOs:1, 3, 5, 7, and 9,and encoding a protein that is functionally equivalent to the proteinconsisting of the amino acid sequence of any one of SEQ ID NOs:2, 4, 6,8, and 10;

(2) a DNA encoding a partial peptide of a protein consisting of theamino acid sequence of any one of SEQ ID NOs:2, 4, 6, 8, and 10;

(3) a protein or peptide that is encoded by the DNA described in (1) or(2);

(4) a vector into which the DNA described in (1) or (2) is inserted;

(5) a transformant harboring the DNA described in (1) or (2), or thevector described in (4);

(6) a method for producing the protein or peptide of (3), comprising thesteps of: culturing said transformant of (5), and recovering theexpressed protein from said transformant or the culture supernatant;

(7) an antibody binding to the protein of (3);

(8) a polynucleotide complementary to either a DNA that comprises thenucleotide sequence of any one of SEQ ID NOs:1, 3, 5, 7, and 9 or itscomplementary strand, wherein the polynucleotide comprises at least 15nucleotides; and,

(9) a method of screening for a compound that binds to the protein of(3), comprising the steps of:

(a) contacting a test sample with said protein or partial peptidethereof;

(b) detecting the binding activity of the test sample with the proteinor partial peptide thereof; and,

(c) selecting the compound that binds to the protein or partial peptidethereof.

The present invention provides a novel hemopoietin receptor “NR12”.According to the results of the database searches on GenBank as well as5′-RACE and 3′-RACE analysis, the present inventors finally succeeded inidentifying and isolating a novel hemopoietin receptor gene NR12. It wasfound that at least three splice variants are transcribed from NR12. Oneof these variants, the cDNA clone NR12.1, encodes a solublereceptor-like protein. The other two predicted to encode transmembranereceptor proteins, cDNA clone NR12.2 and NR12.3, encode a proteinpresumed to have an intracellular region as short as 51 amino acids andas long as 252 amino acids, respectively.

Furthermore, the present inventors conducted PCR cloning against a cDNAlibrary of human thymus to isolate the continuous full-length codingsequences (CDS). A clone having almost the same full-length ORF asNR12.2 was named NR12.4, and that having almost the same full-length ORFas NR12.3 was named NR12.5.

The nucleotide sequence of NR12.1 cDNA is shown in SEQ ID NO:1, and thecorresponding amino acid sequence of the protein encoded by the cDNA isshown in SEQ ID NO:2. The nucleotide sequence of NR12.2 cDNA is shown inSEQ ID NO:3, and the corresponding amino acid sequence of the proteinencoded by the cDNA is shown in SEQ ID NO:4. The nucleotide sequence ofNR12.3 cDNA is shown in SEQ ID NO:5, and the corresponding amino acidsequence of the protein encoded by the cDNA is shown in SEQ ID NO:6. Thenucleotide sequence of NR12.4 cDNA is shown in SEQ ID NO:7, and thecorresponding amino acid sequence of the protein encoded by the cDNA isshown in SEQ ID NO:8. The nucleotide sequence of NR12.5 cDNA is shown inSEQ ID NO:9, and the corresponding amino acid sequence of the proteinencoded by the cDNA is shown in SEQ ID NO:10.

Because the extracellular regions of NR12.1, NR12.2, NR12.3, NR12.4, andNR12.5 are almost identical, these regions are thought to have the sametertiary structure and thereby recognize the same specific ligand.

Analyses of the gene expression in various human organs using RT-PCRrevealed: strong expression of NR12 in hematopoietic cell line tissuesand immune cell line tissues such as adult spleen, thymus, lymph node,bone marrow, and peripheral leukocyte; and expression in testis, liver,lung, kidney, pancreas, and gastrointestinal tract, such as smallintestine and colon. Additionally, expression of NR12 was also observedin all the analyzed mRNA derived from human fetal organs. From therevealed distribution pattern of NR12 gene expression, it was presumedthat NR12 encodes a novel hematopoietic factor receptor, primarilybecause localization of strong expression in tissues thought to includeimmune cell lines and hematopoietic cells was detected. Furthermore, thefact that NR12 expression was observed in tissues other than thosedescribed above suggests that NR12 can regulate not only physiologicalfunctions of the immune system and hematopoietic system in vivo but alsovarious other physiological functions in vivo.

The above NR12 proteins are potentially useful for medical application.Since NR12.1 is expressed in thymus, peripheral leukocytes, and spleen,it is predicted to be a receptor for an unknown hemopoietic factor.Therefore, NR12 proteins are useful tools in the identification of theunknown hemopoietic factor. They may also be used to screen a peptidelibrary or synthetic chemical compounds to isolate or identify agonistsand antagonists that can functionally bind to the NR12 molecule.Moreover, clinical application is expected of novel molecules binding tothe NR12 molecule and specific antibodies that can limit the function ofthe NR12 molecule to regulate the immune response or hematopoiesis invivo, by searching such molecules and antibodies.

NR12 is expected to be expressed in a restricted population of cells inthe hemopoietic tissues, and thus, anti-NR12 antibodies are useful forthe isolation of such cell populations. The isolated cell populationsmay be used in cell transplantation. Furthermore, it is expected thatthe anti-NR12 antibody may be used for the diagnosis or treatment ofdiseases, such as leukemia.

On the other hand, the soluble proteins comprising the extracellulardomain of NR12 protein and the splice variant of NR12, NR12.1, may beused as a decoy-type receptor to inhibit the NR12 ligand. They may beuseful for the treatment of diseases in which NR12 is implicated, suchas leukemia.

The present invention includes proteins that are functionally equivalentto the NR12 protein. For example, homologues of human NR12 protein areincluded. Herein, the term “functionally equivalent” refers to proteinshaving an equivalent biological activity as compared to that of an NR12protein. Such biological activity may include the protein activity as amembrane bound or soluble form hematopoietic factor receptor.

Methods of introducing mutations for preparing proteins that arefunctionally equivalent to another protein are well known to a personskilled in the art. For example, one skilled in the art may usesite-directed mutagenesis (Hashimoto-Gotoh et al., Gene 152:271-275,1995; Zoller et al., Methods Enzymol. 100:468-500, 1983; Kramer et al.,Nucleic Acids Res. 12:9441-9456, 1984; Kramer et al., Methods. Enzymol.154:350-367, 1987; Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492, 1985;Kunkel, Methods Enzymol. 85:2763-2766, 1988) and such in order tointroduce an appropriate mutation into the amino acid sequence of thehuman NR12 protein and prepare a protein that is functionally equivalentto the protein. Mutation of amino acids may occur in nature as well. Theproteins of the present invention includes proteins having the aminoacid sequence of human NR12 protein in which one or more amino acids aremutated, so long as the resulting proteins are functionally equivalentto human NR12 protein.

As a protein functionally equivalent to the NR12 protein of theinvention, the following can be specifically mentioned: one in which oneor two, preferably, two to 30, more preferably, two to 10 amino acidsare deleted in any one of the amino acid sequences of SEQ ID NOs:2, 4,6, 8, or 10; one in which one or two, preferably, two to 30, morepreferably, two to 10 amino acids have been added into any one of theamino acid sequences of SEQ ID NOs:2, 4, 6, 8, or 10; one in which oneor two, preferably, two to 30, more preferably, two to 10 amino acidshave been substituted with other amino acids in any one of the aminoacid sequences of SEQ ID NOs:2, 4, 6, 8, or 10.

As for the amino acid residue to be mutated, it is preferable that it bemutated into a different amino acid that allows the properties of theamino acid side-chain to be conserved. Examples of properties of aminoacid side chains are the following: hydrophobic amino acids (A, I, L, M,F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S,T), and amino acids comprising the following side chains: an aliphaticside-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain(S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acidand amide containing side-chain (D, N, E, Q); a base containingside-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W)(The parenthetic letters indicate the one-letter codes of amino acids).

It is known that a protein may have an amino acid sequence modified bydeletion, addition, and/or substitution of other amino acids for one ormore amino acid residues, yet still retain its biological activity (Market al., Proc. Natl. Acad. Sci. USA 81:5662-5666, 1984; Zoller et al.,Nucleic Acids Res. 10:6487-6500, 1982; Wang et al., Science224:1431-1433; Dalbadie-McFarland et al., Proc. Natl. Acad. Sci. USA79:6409-6413, 1982).

A fusion protein comprising human NR12 protein is an example of aprotein in which one or more amino acids residues have been added to theamino acid sequence of a human NR12 protein (e.g., SEQ ID NO:2, 4 6, 8or 10). A fusion protein is made by fusing the human NR12 protein withanother peptide(s) or protein(s) and is included in the presentinvention. A fusion protein can be prepared by ligating a DNA encodingthe human NR12 protein of the present invention with a DNA encodinganother peptide(s) or protein(s) in frame, introducing the ligated DNAinto an expression vector, and expressing the fusion gene in a host.Methods known by one skilled in the art can be used for preparing such afusion gene. There is no restriction as to the other peptide(s) orprotein(s) that is (are) fused to the protein of the present invention.

Other peptide(s) to be fused with a protein of the present inventioninclude known peptides, for example, FLAG (Hopp et al., Biotechnology6:1204-1210, 1988), 6×His consisting of six His (histidine) residues,10×His, Influenza agglutinin (HA), human c-myc fragment, VSV-GPfragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigenfragment, lck tag, α-tubulin fragment, B-tag, Protein C fragment, and soon. Other examples of proteins to be fused with the protein of thepresent invention are the GST (glutathione-S-transferase), Influenzaagglutinin (HA), immunoglobulin constant region, β-galactosidase, MBP(maltose-binding protein), and such.

Fusion proteins can be prepared by fusing commercially available DNAencoding these peptides or proteins with DNA encoding a protein of thepresent invention and expressing the fused DNA prepared.

The hybridization technique (Sambrook et al., Molecular Cloning 2nd ed.,9.47-9.58, Cold Spring Harbor Lab. Press, 1989) is well known to thoseskilled in the art as an alternative method for preparing a proteinfunctionally equivalent to a certain protein. More specifically, oneskilled in the art can utilize the general procedure to obtain a proteinfunctionally equivalent to a human NR12 protein by isolating DNA havinga high homology with the whole or part of a DNA sequence encoding thehuman NR12 protein (e.g., SEQ ID NO:1, 3, 5, 7 or 9). Thus, the proteinsof the present invention include such proteins, that are encoded by DNAsthat hybridizes with a DNA encoding a human NR12 protein or part thereofand that are functionally equivalent to a human NR12 protein. Examplesinclude homologues of human NR12 in other mammals (for example, those ofmonkey, rat, mouse, rabbit, and bovine gene). In order to isolate a cDNAwith high homology to a DNA encoding a human NR12 protein from animals,it is preferable to use a hematopoietic cell line tissue such as spleen,thymus, lymph node, bone marrow, and peripheral leukocyte; however, theinvention is not limited thereto.

Stringent hybridization conditions for isolating DNA encoding proteinsfunctionally equivalent to a human NR12 protein can be suitably selectedby one skilled in the art, and for example, low-stringent conditions canbe given. Low-stringent conditions are, for example, 42° C., 2×SSC, and0.1% SDS, and preferably, 50° C., 2×SSC, and 0.1% SDS. High stringentconditions are more preferable and include, for example, 65° C., 2×SSC,and 0.1% SDS. Under these conditions, at lower temperatures, the DNAobtained will have a lower homology. Conversely, it is expected that thehomology of the obtained DNA will be higher at higher temperatures.However, several factors other than temperature, such as saltconcentration, can also influence the stringency of hybridization andone skilled in the art can routinely select the factors to accomplish asimilar stringency.

In place of hybridization, the gene amplification method, for example,the polymerase chain reaction (PCR) method can be utilized to isolatethe object DNA, using primers synthesized based on the sequenceinformation of a DNA (e.g., SEQ ID NO:1, 3, 5, 7 or 9) encoding humanNR12 protein.

Proteins that are functionally equivalent to human NR12 protein, encodedby DNA isolated through the above hybridization technique or by the geneamplification techniques, usually have a high homology to the amino acidsequence of the human NR12 protein. The proteins of the presentinvention also include proteins that are functionally equivalent to thehuman NR12 protein, which also have a high homology with the proteincomprising any one of the amino acid sequences of SEQ ID NO:2, 4, 6, 8,and 10. High homology is normally defined as a homology of 70% orhigher, preferably 80% or higher, more preferably 90% or higher, andmost preferably 95% or higher. The homology of a protein can bedetermined by the algorithm in “Wilbur, W. J. and Lipman, D. J. Proc.Natl. Acad. Sci. USA (1983) 80, 726-730”.

The amino acid sequence, molecular weight, isoelectric point, thepresence or absence of sugar chains, and the form of a protein of thepresent invention may differ according to the producing cells, host, orpurification method described below. However, so long as the obtainedprotein has an equivalent function to human NR12 protein (SEQ ID NO:2,4, 6, 8 or 10), it is included in the present invention. For example, ifa protein of the present invention is expressed in prokaryotic cells,such as E. coli, a methionine residue is added at the N-terminus of theamino acid sequence of the expressed protein. If a protein of thepresent invention is expressed in eukaryotic cells, such as mammaliancells, the N-terminal signal sequence is removed. Such proteins are alsoincluded as proteins of the present invention.

For example, as a result of analysis of the protein of the inventionbased on the method in “Von Heijne, G., Nucleic Acids Research, (1986),14, 4683-4690”, it was presumed that the signal sequence extends fromthe 1^(st) Met to the 23^(rd) Gly in the amino acid sequences of SEQ IDNO:2, 4, 6, 8 and 10. Therefore, the present invention encompasses aprotein comprising the sequence from the 24^(th) Gly to 337^(th) Cys inthe amino acid sequence of SEQ ID NO:2. Similarly, the present inventionencompasses a protein comprising the sequence from the 24^(th) Gly to428^(th) Ser in the amino acid sequence of SEQ ID NO:4. Similarly, thepresent invention encompasses a protein comprising the sequence from the24^(th) Gly to 629^(th) Lys in the amino acid sequence of SEQ ID NO:6.Similarly, the present invention encompasses a protein comprising thesequence from the 24^(th) Gly to 428^(th) Ser in the amino acid sequenceof SEQ ID NO:8. Similarly, the present invention encompasses a proteincomprising the sequence from the 24^(th) Gly to 629^(th) Lys in theamino acid sequence of SEQ ID NO:10.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological macromolecules. For example, the substantially purepolypeptide is at least 75%, 80, 85, 95, or 99% pure by dry weight.Purity can be measured by any appropriate standard method known in theart, for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

Accordingly, the invention includes a polypeptide having a sequenceshown as SEQ ID NO:2, 4, 6, 8 or 10. The invention also includes apolypeptide, or fragment thereof, that differs from the correspondingsequence shown as SEQ ID NO:2, 4, 6, 8 or 10. The differences are,preferably, differences or changes at a non-essential residue or aconservative substitution. In one embodiment, the polypeptide includesan amino acid sequence at least about 60% identical to a sequence shownas SEQ ID NO:2, 4, 6, 8 or 10, or a fragment thereof. Preferably, thepolypeptide is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% ormore identical to SEQ ID NO:2, 4, 6, 8 or 10 and has at least onereceptor activity described herein, e.g., a hemopoietin receptoractivity. Preferred polypeptide fragments of the invention are at least10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, or more, of thelength of the sequence shown as SEQ ID NO:2, 4, 6, 8 or 10 and have atleast one receptor activity described herein, e.g., a hemopoietinreceptor activity. Or alternatively, the fragment can be merely animmunogenic fragment.

A protein of the present invention can be prepared by methods known toone skilled in the art, as a recombinant protein, and also as a naturalprotein. A recombinant DNA can be prepared by inserting a DNA encodingthe protein of the present invention (for example, the DNA comprisingthe nucleotide sequence of SEQ ID NO:1, 3, 5, 7 or 9) into a suitableexpression vector, introducing the vector into a suitable host cell, andcollecting the extract from the resulting transformant. After obtainingthe extract, recombinant protein can be purified and prepared bysubjecting to chromatography, such as ion exchange chromatography,reverse phase chromatography, gel filtration, and such, or affinitychromatography, wherein antibodies against the protein of the presentinvention are immobilized, or using one or more of these columns incombination.

Further, when a protein of the present invention is expressed withinhost cells (for example, animal cells and E. coli), as a fusion proteinwith glutathione-S-transferase protein or as a recombinant proteinsupplemented with multiple histidines, the expressed recombinant proteincan be purified using a glutathione column or nickel column.

After purifying the fusion protein, it is also possible to excluderegions other than the objective protein by cutting with thrombin,factor-Xa, and such, as required.

A natural protein may be isolated by methods known to one skilled in theart. For example, extracts of tissue or cells expressing a protein ofthe invention may be reacted with an affinity column described below, towhich antibodies binding to the human NR12 protein are attached, toisolate the natural protein. Polyclonal or monoclonal antibodies may beused.

The present invention also includes partial peptides of the proteins ofthe present invention. A partial peptide consists of an amino acidsequence specific to a protein of the present invention and is composedof at least 7 amino acids, preferably more than 8 amino acids, and morepreferably more than 9 amino acids. The partial peptides may be useful,for example, for preparing antibodies against a protein of the presentinvention; for screening compounds binding to a protein of the presentinvention, or for screening accelerators or inhibitors of a protein ofthe present invention. Alternatively, they may be used as antagonistsfor the ligand of a protein of the present invention. A partial peptideof a protein of the present invention is, for example, a partial peptidehaving the active center of the protein consisting of the amino acidsequences of SEQ ID NO:2, 4, 6, 8, or 10. Additionally, the partialpeptides may comprise one or more regions of the hydrophilic region andhydrophobic region determined by hydrophobicity plot analysis. Thesepartial peptides may contain the whole hydrophilic region or a part of ahydrophilic region, or may contain the whole or a part of thehydrophobic region. Moreover, for example, soluble proteins and proteinscomprising extracellular regions of a protein of the invention are alsoencompassed in the invention.

The partial peptides of the invention may be produced by geneticengineering techniques, well-known peptide synthesizing methods, or byexcising a protein of the invention with a suitable peptidase. Forexample, the solid phase synthesizing method or liquid phasesynthesizing method may be used as peptide synthesizing method.

Another object of the present invention is to provide a DNA encoding aprotein of the present invention. The DNA may be useful for producingthe above proteins of the present invention in vivo or in vitro.Furthermore, for example, it is also possible to use the DNA forapplication to gene therapy and such of diseases arising fromabnormalities of the gene encoding the protein of the present invention.The DNA may be provided in any form, so long as it encodes a protein ofthe present invention. Thus, the DNA may be a cDNA synthesized frommRNA, genomic DNA, or chemically synthesized DNA. Furthermore, a DNAcomprising any nucleotide sequence based on the degeneracy of geneticcode may be included so long as it encodes a protein of the presentinvention.

As used herein, an “isolated nucleic acid” is a nucleic acid, thestructure of which is not identical to that of any naturally occurringnucleic acid or to that of any fragment of a naturally occurring genomicnucleic acid spanning more than three genes. The term therefore covers,for example, (a) a DNA which has the sequence of part of a naturallyoccurring genomic DNA molecule but is not flanked by both of the codingsequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. Specifically excluded from this definition are nucleicacids present in random, uncharacterized mixtures of different DNAmolecules, transfected cells, or cell clones, e.g., as these occur in aDNA library such as a cDNA or genomic DNA library.

Accordingly, in one aspect, the invention provides an isolated orpurified nucleic acid molecule that encodes a polypeptide describedherein or a fragment thereof. Preferably, the isolated nucleic acidmolecule includes a nucleotide sequence that is at least 60% identicalto the nucleotide sequence shown in SEQ ID NO:1, 3, 5, 7 or 9. Morepreferably, the isolated nucleic acid molecule is at least 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, identical to the nucleotide sequence shown in SEQ ID NO:1, 3, 5, 7or 9. In the case of an isolated nucleic acid molecule which is longerthan or equivalent in length to the reference sequence, e.g., SEQ IDNO:1, 3, 5, 7 or 9, the comparison is made with the full length of thereference sequence. Where the isolated nucleic acid molecule is shorterthat the reference sequence, e.g., shorter than SEQ ID NO:1, 3, 5, 7 or9, the comparison is made to a segment of the reference sequence of thesame length (excluding any loop required by the homology calculation).

As used herein, “% identity” of two amino acid sequences, or of twonucleic acid sequences, is determined using the algorithm of Karlin andAltschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as inKarlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993).Such an algorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotidesearches are performed with the NBLAST program, score=100,wordlength=12. BLAST protein searches are performed with the XBLASTprogram, score=50, wordlength=3. To obtain gapped alignment forcomparison purposes GappedBLAST is utilized as described in Altschul etal. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST andGappedBLAST programs the default parameters of the respective programs(e.g., XBLAST and NBLAST) are used to obtain nucleotide sequenceshomologous to a nucleic acid molecule of the invention.

The DNA of the present invention can be prepared by any method known toa person skilled in the art. For example, the DNA of the presentinvention may be prepared by constructing a cDNA library from cellsexpressing the protein of the present invention, and conductinghybridization using as a probe a partial sequence of a DNA of thepresent invention (for example, SEQ ID NO:1, 3, 5, 7 or 9). A cDNAlibrary may be constructed, for example, according to the methoddescribed in the literature (Sambrook et al., Molecular Cloning, ColdSpring Harbor Laboratory Press, 1989), or a commercial cDNA library maybe used. Alternatively, the DNA may be prepared by obtaining RNA from acell expressing a protein of the present invention, synthesizing oligoDNA based on the sequence of a DNA of the present invention (forexample, SEQ ID NO:1, 3, 5, 7 or 9), conducting PCR using thesynthesized DNA as primers, and amplifying the cDNA encoding a proteinof the present invention.

By determining the nucleotide sequence of the obtained cDNA, thetranslation region encoded by the cDNA can be determined, and the aminoacid sequence of the protein of the present invention can be obtained.Furthermore, genomic DNA can be isolated by screening genomic DNAlibraries using the obtained cDNA as a probe.

Specifically, this can be done as follows: first, mRNA is isolated fromcells, tissues, or organs expressing a protein of the invention (forexample, hematopoietic-competent cell line tissue such as spleen,thymus, lymph node, bone marrow, peripheral leukocyte andimmunocompetent cell line tissue, and such). To isolate the mRNA, atfirst, whole RNA is prepared using well-known methods, for example, theguanidine ultracentrifugation method (Chirgwin et al., Biochemistry18:5294-5299, 1979), the AGPC method (Chomczynski et al., Anal. Biochem.162:156-159, 1987), and such. Next, mRNA from whole mRNA can be purifiedusing the mRNA Purification Kit (Pharmacia), and such. Alternatively,mRNA may be directly prepared by QuickPrep mRNA Purification Kit(Pharmacia).

cDNA can then be synthesized using reverse transcriptase from theobtained mRNA. cDNA can be synthesized by using the AMV ReverseTranscriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo), etc.Additionally, cDNA synthesis and amplification may be also performedusing the primer and such described herein, following the 5′-RACE method(Frohman et al. Proc. Natl. Acad. Sci. USA 85:8998-9002, 1988; Belyavskyet al., Nucleic Acids Res. 17:2919-2932, 1989) utilizing the polymerasechain reaction (PCR) and the 5′-Ampli FINDER RACE Kit (Clontech).

The objective DNA fragment is prepared from the obtained PCR product andligated with a vector DNA. Thus, a recombinant vector is created andintroduced into E. coli, and such, and colonies are selected to preparethe desired recombinant vector. The nucleotide sequence of the objectiveDNA can be verified by conventional methods, for example,dideoxynucleotide chain termination.

With regards to the DNA of the invention, a sequence with higherexpression efficiency can be designed by considering the codon usagefrequency in the host used for the expression (Grantham et al., NucleicAcids Res. 9:43-74, 1981). The DNA of the present invention may also bemodified using commercially available kits and conventional methods.Illustrative modifications include, for instance, digestion byrestriction enzymes, insertion of synthetic oligonucleotides andsuitable DNA fragments, addition of linkers, insertion of a initiationcodon (ATG) and/or stop codon (TAA, TGA, or TAG), and such.

Specifically, the DNA of the present invention includes DNA comprisingthe nucleotide sequence from the 98^(th) “A” to the 1108^(th) “C” of SEQID NO:1; the 98^(th) “A” to 1381^(st) “C” of SEQ ID NO:3; the 98^(th)“A” to 1984^(th) “G” of SEQ ID NO:5; the 1^(st) “A” to 1284^(th) “C” ofSEQ ID NO:7; and the 1^(st) “A” to 1887^(th) “G” of SEQ ID NO:9.

Furthermore, the present invention includes DNA that hybridize understringent conditions to the DNA consisting of any one of the nucleotidesequence of SEQ ID NO:1, 3, 5, 7 or 9, so long as the resulting DNAencodes a protein functionally equivalent to the above-mentioned proteinof the invention.

One skilled in the art can suitably select stringent conditions, and forexample, low-stringent conditions can be given. Low-stringent conditionsare, for example, 42° C., 2×SSC, and 0.1% SDS, and preferably 50° C.,2×SSC, and 0.1% SDS. More preferable are highly stringent conditionswhich are, for example, 65° C., 2×SSC, and 0.1% SDS. Under theseconditions, the higher the temperature, the higher the homology of theobtained DNA will be. The above hybridizing DNA is preferably a naturalDNA, such as cDNA and chromosomal DNA.

Moreover, the present invention provides a vector containing a DNA ofthe present invention as an insert. The vector of the present inventionmay be useful for maintaining the DNA of the present invention in hostcells or producing the protein of the present invention.

If the host cell is E. coli (such as JM109, DH5α, HB101, and XL1Blue),any vector may be used as long as it contains the “ori” foramplification in E. coli that enables large-scale preparation, and aselection marker for transformants (for example, a drug-resistance genethat enables selection by a drug such as ampicillin, tetracycline,kanamycin, and chloramphenicol). For example, M13-series vectors,pUC-series vectors, pBR322, pBluescript, pCR-Script, and so on can beused. For the purpose of subcloning or excision of a cDNA, pGEM-T,pDIRECT, pT7, and such may be used as well. For producing the protein ofthe present invention, an expression vector is especially useful. Forexample, if the protein is to be expressed in E. coli, the expressionvector must have characteristics such as those mentioned above to beamplified in E. coli. Additionally, when E. coli, such as JM109, DH5α,HB101, or XL1 Blue, is used as the host cell, the vector must have apromoter, for example, the lacZ promoter (Ward et al., Nature341:544-546, 1989; FASEB J. 6:2422-2427, 1992), the araB promoter(Better et al., Science 240:1041-1043, 1988), the T7 promoter, and such,that can efficiently express the desired gene in E. coli. Such vectorsinclude pGFX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP, pET(in this case, a host is preferably BL21 which expresses T7 RNApolymerase), and so on, except those mentioned above.

Vectors may be introduced into host cells, for example, by the calciumchloride method or electroporation. The vector may also contain a signalsequence for polypeptide secretion. The pe1B signal sequence (Lei etal., J. Bacteriol. 169:4379, 1987) may be used to produce the proteinsin the periplasm in E. coli.

For example, an expression vector for the preparation of a protein ofthe present invention may be a mammal-derived expression vector (forexample, pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids. Res. 18(17):5322,1990), pEF, and pCDM8); an insect cell-derived expression vector (forexample, “Bac-to-BAC baculovirus expression system” (GIBCO BRL),pBacPAK8); a plant-derived expression vector (for example, pMH1 andpMH2); an animal virus-derived expression vector (for example, pHSV,pMV, and pAdexLcw); a retrovirus-derived expression vector (for example,pZIpneo); an yeast-derived expression vector (for example, “PichiaExpression Kit” (Invitrogen), pNV11, and SP-Q01); or a Bacillussubtilis-derived expression vectors (for example, pPL608 and pKTH50),other than E. coli.

For the expression in animal cells, such as CHO, COS, and NIH3T3 cells,the expression vector must have a promoter such as the SV40 promoter(Mulligan et al., Nature 277:108, 1979), MMLV-LTR promoter, the EF1αpromoter (Mizushima et al., Nucleic Acids Res. 18:5322, 1990), and theCMV promoter. More preferably, the vector may contain a marker gene forthe selection of transformants (for example, a drug resistance gene forselection by a drug such as neomycin and G418). Such vectors includepMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOp13, and so on.

Furthermore, in order to achieve stable gene expression andamplification of the copy number of genes in cell, CHO cells deficientin the metabolic pathway for nucleotide synthesis may be used. The CHOcell is transfected with an expression vector comprising the DHFR genethat complements the deficiency (for example, pCHO I), then the vectormay be amplified by methotrexate (MTX) treatment. For transient geneexpression, COS cells containing a gene expressing the SV40 T-antigen onits chromosome may be used to transform with a vector containing theSV40 replication origin (e.g., pcD). Examples of replication origins tobe used in the present invention include those derived frompolyomavirus, adenovirus, bovine papilomavirus (BPV), and such.Moreover, to amplify the gene copies in host cell lines, the expressionvector may include an aminoglycoside transferase (APH) gene, thymidinekinase (TK) gene, E. coli xanthine guanine phosphoribosyl transferase(Ecogpt) gene, dihydrofolate reductase (dhfr) gene, and such as aselective marker.

On the other hand, in vivo expression of a DNA of the present inventionin animals may be performed by, for example, by inserting a DNA of thepresent invention into an appropriate vector and introducing the vectorinto the body using retrovirus, liposome, cationic liposome, adenovirus,and so on. It is possible to use these methods to perform gene therapyfor diseases that arise from mutations in the NR12 gene of the presentinvention. Examples of vectors used for this purpose include, forexample, adenovirus vectors (for example pAdexlcw), retrovirus vectors(for example, pZIPneo), and such, but are not limited thereto. Generalgene manipulations, for example, insertion of the DNA of the presentinvention into a vector, may be performed by using standard methods(Molecular Cloning, 5.61-5.63). The vector may be administered to aliving body through ex vivo or in vivo methods.

Another object of the present invention is to provide a transformantthat contains a DNA or vector of the present invention. The host cell toinsert a vector of the invention is not limited in any way, and forexample, E. coli, a variety of animal cells, and so on may be used. Thehost cells of the present invention may be, for example, used as aproduction system for preparing or expressing a protein of the presentinvention. In vitro and in vivo production systems are known asproduction system for producing proteins. Production systems usingeukaryotic cells and prokaryotic cells may be used as the in vitroproduction systems.

When using eukaryotic cells, production system using, for example,animal cells, plant cells, and fungal cells are available as hosts.Exemplary animal cells to be used include mammalian cells such as CHO(J. Exp. Med. 108:945, 1995), COS, 3T3, myeloma, baby hamster kidney(BHK), HeLa, Vero cells; amphibian cells such as Xenopus oocytes (Valleet al. Nature 291:338-340, 1981); and insect cells such as Sf9, Sf21, orTn5. As CHO cells, especially DHFR gene-deficient CHO cell, dhfr-CHO(Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980), and CHO K-1 (Proc.Natl. Acad. Sci. USA 60:1275, 1968) can be suitably used. Forlarge-scale preparation in animal cells, CHO cells may be preferablyused. The vector may be introduced into host cells, for example, by thecalcium phosphate method, the DEAE dextran method, methods usingcationic liposome DOTAP (Boehringer Mannheim), the electroporationmethod, the lipofection method, and so on.

Nicotiana tabacum-derived cells are well known as protein productionsystems in plant cells, and these can be callus cultured. As fungalcells, yeasts such as the Saccharomyces genus, for example,Saccharomyces cerevisiae; filamentous fungi, such as Aspergillus genus,for example, Aspergillus niger are known.

Bacterial cells may be used as prokaryotic production system. Asbacterial cells, E. coli, for example, JM109, DH5α, HB101, and such, aswell as others like Bacillus subtilis are known.

Proteins can be obtained by transforming these cells with the objectiveDNA, and culturing the transformed cells in vitro. Transformants can becultured according to known methods. For example, DMEM, MEM, RPMI1640,and IMDM can be used as culture media of animal cells. Occasionally,fetal calf serum (FCS) and such serum supplements may be added in theabove media; alternatively, a serum-free culture medium may be used. ThepH of the culture medium is preferably from about pH 6 to 8. Theculturing is usually performed at about 30 to 40° C., for about 15 to200 hr, and the culture medium changes, aeration, and stirring are doneas necessary.

On the other hand, for example, production systems using animals andplants may be given as in vivo protein production systems. The objectiveDNA is introduced into the animal or plant, and the protein is producedwithin the plant or animal, and then, the protein is recovered. The term“host” as used in the present invention encompasses such animals andplants as well.

When using animals, mammalian and insect production systems can be used.As mammals, goats, pigs, sheep, mice, and bovines may be used (VickiGlaser, SPECTRUM Biotechnology Applications, 1993). Alternatively,transgenic animals may also be used when using mammals.

For instance, the objective DNA may be prepared as a fusion gene with agene encoding a protein intrinsically produced into milk, such as goat βcasein. Next, the DNA fragment comprising the fusion gene is injectedinto goat's embryo, and this embryo is implanted in female goat. Theobjective protein can be recovered from the milk of the transgenic goatsproduced from the goat that received the embryo and offspring thereof.To increase the amount of protein-containing milk produced from thetransgenic goat, a suitable hormone(s) may be administered to thetransgenic goats (Ebert et al., Bio/Technology 12:699-702, 1994).

Silk worms may be used as insects. When using silk worms, they areinfected with baculoviruses to which the DNA encoding objective proteinhas been inserted, and the desired protein can be recovered from bodyfluids of the silk worm (Susumu et al., Nature 315:592-594, 1985).

When using plants, for example, tobacco can be used. In the case oftobacco, the DNA encoding the objective protein is inserted into a plantexpression vector, for example, pMON530, and this is inserted into abacteria, such as Agrobacterium tumefaciens. This bacterium is infectedto tobacco, for example, Nicotiana tabacum, and it is able to obtain thedesired polypeptide from the tobacco leaves (Julian et al., Eur. J.Immunol. 24:131-138, 1994).

Thus-obtained proteins of the present invention are isolated from insideor outside (e.g., medium) of the host cell, and may be purified as asubstantially pure homogeneous protein. The separation and purificationof the protein can be done using conventional separation andpurification methods used to purify proteins and are not limited to anyspecific method. For instance, column chromatography, filter,ultrafiltration, salt precipitation, solvent precipitation, solventextraction, distillation, immunoprecipitation, SDS-polyacrylamide gelelectrophoresis, isoelectric point electrophoresis, dialysis,recrystallization, and such may be suitably selected, or combined toseparate and purify the protein.

For example, affinity chromatography, ion-exchange chromatography,hydrophobic chromatography, gel filtration, reverse phasechromatography, adsorption chromatography, and such can be exemplifiedas chromatographies (Strategies for Protein Purification andCharacterization: A Laboratory Course Manual. Ed. Daniel R. Marshak etal., Cold Spring Harbor Laboratory Press, 1996). These chromatographiescan be performed by liquid chromatography, such as HPLC, FPLC, and such.The present invention encompasses proteins highly purified by using suchpurification methods.

Proteins can be arbitrarily modified, or peptides can be partiallyexcised by treating the proteins with appropriate protein modificationenzymes prior to or after the purification. Trypsin, chymotrypsin,lysyl-endopeptidase, protein kinase, glucosidase, and such are used asprotein modification enzymes.

The present invention also provides antibodies that bind to the proteinof the invention. There is no particular restriction as to the form ofthe antibody of the invention and the present invention includespolyclonal antibodies, as well as monoclonal antibodies. The antiserumobtained by immunizing animals such as rabbits with a protein of theinvention, as well polyclonal and monoclonal antibodies of all classes,human antibodies, and humanized antibodies produced by geneticrecombination, are also included.

A protein of the invention that is used as a sensitizing antigen forobtaining antibodies is not restricted by the animal species from whichit is derived, but is preferably a protein derived from mammals, forexample, humans, mice, or rats, especially preferably from humans.Protein of human origin can be obtained by using the nucleotide sequenceor amino acid sequences disclosed herein.

Herein, an intact protein or its partial peptide may be used as theantigen for immunization. As partial peptides of the proteins, forexample, the amino (N)-terminal fragment of the protein, and the carboxy(C)-terminal fragment can be given. “Antibody” as used herein means anantibody that specifically reacts with the full-length or fragments ofthe protein.

A gene encoding a protein of the invention or a fragment thereof isinserted into a known expression vector, and, by transforming the hostcells with the vector described herein, the desired protein or afragment thereof is recovered from outside or inside the host cellsusing standard methods. This protein can be used as the sensitizingantigen. Also, cells expressing the protein, cell lysates, or achemically synthesized protein of the invention may be also used as asensitizing antigen.

The mammal that is immunized by the sensitizing antigen is notrestricted; however, it is preferable to select animals by consideringthe compatibility with the parent cells used in cell fusion. Generally,animals belonging to the rodentia, lagomorpha, and Primates are used.

Examples of animals belonging to rodentia that may be used include, forexample, mice, rats, hamsters, and such. Examples of animals belongingto lagomorpha that may be used include, for example, rabbits. Examplesof animals of Primates that may be used include, for example, monkeys.Examples of monkeys to be used include the infraorder catarrhini (oldworld monkeys), for example, Macaca fascicularis, rhesus monkeys, sacredbaboons, chimpanzees, and such.

Well-known methods may be used to immunize animals with the sensitizingantigen. For example, the sensitizing antigen is injectedintraperitoneally or subcutaneously into mammals. Specifically, thesensitizing antigen is suitably diluted and suspended in physiologicalsaline, phosphate-buffered saline (PBS), and so on, and mixed with asuitable amount of general adjuvant if desired, for example, withFreund's complete adjuvant. Then, the solution is emulsified andinjected into the mammal. Thereafter, the sensitizing antigen suitablymixed with Freund's incomplete adjuvant is preferably given severaltimes every 4 to 21 days. A suitable carrier can also be used whenimmunizing and animal with the sensitizing antigen. After theimmunization, the elevation in the level of serum antibody is detectedby usual methods.

Polyclonal antibodies against the proteins of the present invention canbe prepared as follows. After verifying that the desired serum antibodylevel has been reached, blood is withdrawn from the mammal sensitizedwith antigen. Serum is isolated from this blood using conventionalmethods. The serum containing the polyclonal antibody may be used as thepolyclonal antibody, or according to needs, the polyclonalantibody-containing fraction may be further isolated from the serum. Forinstance, a fraction of antibodies that specifically recognize theprotein of the invention may be prepared by using an affinity column towhich the protein is coupled. Then, the fraction may be further purifiedby using a Protein A or Protein G column in order to prepareimmunoglobulin G or M.

To obtain monoclonal antibodies, after verifying that the desired serumantibody level has been reached in the mammal sensitized with theabove-described antigen, immunocytes are taken from the mammal and usedfor cell fusion. For this purpose, splenocytes can be mentioned aspreferable immunocytes. As parent cells fused with the aboveimmunocytes, mammalian myeloma cells are preferably used. Morepreferably, myeloma cells that have acquired the feature, which can beused to distinguish fusion cells by agents, are used as the parent cell.

The cell fusion between the above immunocytes and myeloma cells can beconducted according to known methods, for example, the method byMilstein et al. (Galfre et al., Methods Enzymol. 73:3-46, 1981).

The hybridoma obtained from cell fusion is selected by culturing thecells in a standard selection medium, for example, HAT culture medium(medium containing hypoxanthine, aminopterin, and thymidine). Theculture in this HAT medium is continued for a period sufficient enoughfor cells (non-fusion cells) other than the objective hybridoma toperish, usually from a few days to a few weeks. Then, the usual limitingdilution method is carried out, and the hybridoma producing theobjective antibody is screened and cloned.

Other than the above method for obtaining hybridomas, by immunizing ananimal other than humans with the antigen, a hybridoma producing theobjective human antibodies having the activity to bind to proteins canbe obtained by the method of sensitizing human lymphocytes, for example,human lymphocytes infected with the EB virus, with proteins,protein-expressing cells, or lysates thereof in vitro and fusing thesensitized lymphocytes with myeloma cells derived from human, forexample, U266, having a permanent cell division ability (UnexaminedPublished Japanese Patent Application (JP-A) No. Sho 63-17688).

The monoclonal antibodies obtained by transplanting the obtainedhybridomas into the abdominal cavity of a mouse and extracting ascitescan be purified by, for example, ammonium sulfate precipitation, proteinA or protein G column, DEAE ion exchange chromatography, an affinitycolumn to which the protein of the present invention is coupled, and soon. An antibody of the present invention may be used for thepurification or detection of a protein of the present invention. It mayalso be a candidate as an agonist or antagonist of a protein of thepresent invention. Furthermore, it is possible to use it in antibodytreatment for diseases in which the protein is implicated. For theadministration to human body (antibody treatment), human antibodies orhumanized antibodies are preferably used because of their reducedimmunogenicity.

For example, a human antibody against a protein can be obtained usinghybridomas made by fusing myeloma cells with antibody-producing cellsobtained by immunizing a transgenic animal comprising a repertoire ofhuman antibody genes with an antigen such as protein, protein-expressingcells, or lysates thereof (see WO92-03918, WO93-2227, WO94-02602,WO94-25585, WO96-33735, and WO96-34096).

Other than producing antibodies using hybridoma, antibody producingimmunocytes, such as sensitized lymphocytes that are immortalized byoncogenes, may also be used.

Such monoclonal antibodies can be also obtained as recombinantantibodies produced by using the genetic engineering technique (see, forexample, Borrebaeck C. A. K. and Larrick, J. W., THERAPEUTIC MONOCLONALANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD(1990)). Recombinant antibodies are produced by cloning the encoding DNAfrom immunocytes, such as hybridoma or antibody-producing sensitizedlymphocytes, incorporating into a suitable vector, and introducing thisvector into a host to produce the antibody. The present inventionencompasses such recombinant antibodies as well.

Moreover, the antibody of the present invention may be an antibodyfragment or modified-antibody, so long as it binds to a protein of theinvention. For instance, Fab, F (ab′)₂, Fv, or single chain Fv (scFv) inwhich the H chain Fv and the L chain Fv are suitably linked by a linker(Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988) can begiven as antibody fragments. Specifically, antibody fragments aregenerated by treating antibodies with enzymes, for example, papain orpepsin. Alternatively, they may be generated by constructing a geneencoding an antibody fragment, introducing this into an expressionvector, and expressing this vector in suitable host cells (see, forexample, Co et al., J. Immunol. 152:2968-2976, 1994; Better et al.,Methods Enzymol. 178:476-496, 1989; Pluckthun et al., Methods Enzymol.178:497-515, 1989; Lamoyi, Methods Enzymol. 121:652-663, 1986; Rousseauxet al., Methods Enzymol. 121:663-669, 1986; Bird et al., TrendsBiotechnol. 9:132-137, 1991).

As modified antibodies, antibodies bound to various molecules, such aspolyethylene glycol (PEG), can be used. The antibodies of the presentinvention encompass such modified antibodies as well. To obtain such amodified antibody, chemical modifications are done to the obtainedantibody. These methods are already established and conventional in thefield.

An antibody of the present invention may be obtained as a chimericantibody, comprising non-human antibody-derived variable region andhuman antibody-derived constant region, or as a humanized antibodycomprising non-human antibody-derived complementarily determining region(CDR), human antibody-derived framework region (FR), and humanantibody-derived constant region by using conventional methods.

Antibodies thus obtained can be purified to uniformity. The separationand purification methods used in the present invention for separatingand purifying the antibody may be any method usually used for proteins.For example, column chromatography, such as affinity chromatography,filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gelelectrophoresis, isoelectric focusing, and others, may be appropriatelyselected and combined to isolate and purify the antibodies (Antibodies:A Laboratory Manual. Ed Harlow and David Lane, Cold Spring HarborLaboratory, 1988); however, the invention is not limited thereto.Antibody concentration of the above mentioned antibody can be assayed bymeasuring the absorbance, or by the enzyme-linked immunosorbent assay(ELISA), etc.

Protein A or Protein G column can be used for the affinitychromatography. Protein A column may be, for example, Hyper D, POROS,Sepharose F. F. (Pharmacia), etc.

Other chromatography may also be used, for example, such as ion-exchangechromatography, hydrophobic chromatography, gel filtration,reverse-phase chromatography, and adsorption chromatography (Strategiesfor Protein Purification and Characterization: A Laboratory CourseManual. Ed Daniel R. Marshak et al., Cold Spring Harbor LaboratoryPress, 1996). These may be performed on liquid-phase chromatography suchas HPLC, FPLC, and so on.

Examples of methods that assay the antigen-binding activity of theantibodies of the invention include, for example, measurement ofabsorbance, enzyme-linked immunosorbent assay (ELISA), enzymeimmunoassay (EIA), radioimmunoassay (RIA), and/or immunofluorescence.For example, when using ELISA, a protein of the invention is added to aplate coated with the antibodies of the present invention, and then, theobjective antibody sample, for example, culture supernatants ofantibody-producing cells, or purified antibodies are added. Then,secondary antibody recognizing the primary antibody, which is labeled byalkaline phosphatase and such enzymes, is added, the plate is incubatedand washed, and the absorbance is measured to evaluate theantigen-binding activity after adding an enzyme substrate such asp-nitrophenyl phosphate. As the protein, a protein fragment, forexample, a fragment comprising a C-terminus, or a fragment comprising anN-terminus may be used. To evaluate the activity of the antibody of theinvention, BIAcore (Pharmacia) may be used.

By using these methods, the antibody of the invention and a samplepresumed to contain a protein of the invention are contacted, and theprotein of the invention is detected or assayed by detecting or assayingthe immune complex formed between the above-mentioned antibody and theprotein.

A method of detecting or assaying a protein of the invention is usefulin various experiments using proteins as it can specifically detect orassay the proteins.

Another object of this invention is to provide a polynucleotide of atleast 15 nucleotides that is complementary to the DNA encoding humanNR12 protein (SEQ ID NO:1, 3, 5, 7 or 9) or its complementary strand.

Herein, the term “complementary strand” is defined as one strand of adouble strand polynucleotide composed of A:T and G:C base pairs to theother strand. Also, “complementary” is defined as not only thosecompletely matching within a continuous region of at least 15nucleotides, but also having a homology of at least 70%, preferably 80%,more preferably 90%, and most preferably 95% or higher within thatregion. The homology may be determined using the algorithm describedherein.

Probes and primers for detection or amplification of the DNA encoding aprotein of the invention, or a nucleotide or nucleotide derivative forthe suppression of the protein expression (such as, antisenseoligonucleotide and ribozyme) are included in these polynucleotides.Such polynucleotides may be also used for preparing DNA chips.

The antisense oligonucleotides that hybridize with a portion of thenucleotide sequence of any of SEQ ID NO:1, 3, 5, 7, and 9 are alsoincluded in the antisense oligonucleotides of the present invention.These antisense oligonucleotides are preferably directed against asequence which contains at least 15 continuous nucleotides comprised inany one of the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, and 9. Morepreferably, it is the antisense oligonucleotide against at least 15continuous nucleotides containing a translation start codon.

Derivatives or modified products of antisense oligonucleotides can beused as antisense oligonucleotides. Examples of such modified productsinclude, for example, lower alkyl phosphonate modifications such asmethyl-phosphonate-type or ethyl-phosphonate-type; phosphorothioatemodifications; phosphoroamidate modifications, and such.

The term “antisense oligonucleotides” as used herein means, not onlythose in which the nucleotides corresponding to those constituting aspecified region of a DNA or mRNA are entirely complementary, but alsothose having a mismatch of one or more nucleotides, so long as the DNAor mRNA and the oligonucleotide can specifically hybridize with thenucleotide sequence of SEQ ID NO:1, 3, 5, 7 or 9.

The antisense oligonucleotide derivatives of the present invention actupon cells producing a protein of the invention by binding to the DNA orRNA encoding the protein to inhibit its transcription or translation,and to promote the degradation of the mRNA, and have an effect ofsuppressing the function of the protein of the invention by suppressingthe expression of the protein.

An antisense oligonucleotide derivative of the present invention can bemade into an external preparation, such as a liniment or a poultice, bymixing with a suitable base material, which is inactive against thederivatives.

Also, as needed, the derivatives can be formulated into tablets,powders, granules, capsules, liposome capsules, injections, solutions,nose-drops, freeze-dried agents, and such by adding excipients, isotonicagents, solubilizing agents, stabilizers, preservative substance,pain-killers, and such. These can be prepared using conventionalmethods.

An antisense oligonucleotide derivative is given to the patient bydirectly applying it onto the ailing site, by injecting it into theblood vessel and such, so that it will reach the ailing site. Anantisense-mounting medium can also be used to increase durability andmembrane-permeability. Examples are, liposome, poly-L-lysine, lipid,cholesterol, lipofectin or derivatives of them.

The dosage of the antisense oligonucleotide derivative of the presentinvention can be adjusted suitably according to the patient's conditionand used in desired amounts. For example, a dose range of 0.1 to 100mg/kg, preferably 0.1 to 50 mg/kg can be administered.

The antisense oligonucleotide of the present invention is useful ininhibiting the expression of the protein of the invention, and thereforeis useful in suppressing the biological activity of the proteins of theinvention. Also, expression-inhibitors comprising the antisenseoligonucleotide of the invention are useful, because of their capabilityto suppress the biological activity of the proteins of the invention.

Proteins of this invention are useful in screening for compounds thatbind to the protein. That is, the proteins are used in a method ofscreening for compounds that bind to the proteins of this invention, inwhich the method comprises bringing proteins of this invention intocontact with a test sample that is expected to contain a compound thatmay bind to the proteins and selecting the compound with the activity ofbinding to the proteins of the invention.

Proteins of this invention to be used in the screening of the inventionmay be any of recombinant, natural, or partial peptides. Also, they maybe in the form of proteins expressed on the cell surface or membranefractions. Test samples to be used in the screening method of thepresent invention are not limited, but may be, for example, cellextracts, cell culture supernatants, microbial fermentation products,extracts of marine organisms, plant extracts, purified or partlypurified proteins, peptides, non-peptide compounds, synthetic lowmolecular compounds, or natural compounds. The proteins of thisinvention may be exposed to the sample as purified protein or solubleproteins, in a form bound to a carrier, as fusion proteins with anotherprotein, in a form expressed on the cell surface, or as membranefractions.

A protein of the present invention may be used to screen for otherproteins that bind to the target protein (such as ligands) using avariety of methods known to one skilled in the art. These screeningprocesses can be carried out, for example, by the immunoprecipitationmethod. Specifically, the method can be carried out as follows. The geneencoding a protein of the present invention is expressed by insertingthe gene into an expression vector for foreign gene expression likepSV2neo, pcDNA I, pCD8, and such, and expressing the gene in animalcells, etc. Any generally used promoters may be employed for theexpression, including the SV40 early promoter (Rigby in Williamson(ed.), Genetic engineering, Vol. 3. Academic Press, London, p. 83-141,1982), the EF-1α promoter (Kim et al., Gene 91:217-223, 1990), the CAGpromoter (Niwa et al., Gene 108:193-200, 1991), the RSV LTR promoter(Cullen, Methods in Enzymology 152:684-704, 1987), the SRo: promoter(Takebe et al., Mol. Cell. Biol. 8:466, 1988), the CMV immediate earlypromoter (Seed et al., Proc. Natl. Acad. Sci. USA 84:3365-3369, 1987),the SV40 late promoter (Gheysen et al., J. Mol. Appl. Genet. 1:385-394,1982), the Adenovirus late promoter (Kaufman et al., Mol. Cell. Biol.9:946, 1989), the HSV TK promoter, and soon.

Transfer of a foreign gene into animal cells for its expression can beperformed by any of the following methods, including the electroporationmethod (Chu et al., Nucl. Acid Res. 15:1311-1326, 1987), the calciumphosphate method (Chen et al., Mol. Cell. Biol. 7:2745-2752, 1987), theDEAE dextran method (Lopata et al., Nucl. Acids Res. 12:5707-5717, 1984;Sussman et al., Mol. Cell. Biol. 4:1642-1643, 1985), the lipofectinmethod (Derijard, Cell 7:1025-1037, 1994; Lamb et al., Nature Genetics5:22-30, 1993; Rabindran et al., Science 259:230-234, 1993), and such. Aprotein of the present invention can be expressed as a fusion proteinhaving the recognition site (epitope) for a monoclonal antibody byintroducing a recognition site (epitope) for a monoclonal antibody, thespecificity of which has been established, into the N- or C-terminus ofthe protein of the present invention. For this purpose, a commercialepitope-antibody system can be utilized (Exp. Med. 13:85-90, 1995).Vectors are commercially available which are capable of expressingfusion proteins with β-galactosidase, maltose binding protein,glutathione S-transferase, green florescence protein (GFP), and such,via the multi-cloning site.

To minimize the alteration of the properties of a protein of thisinvention arising from the formation into a fusion protein, a the fusionprotein may be prepared by introducing only a small epitope portioncomprising several to ten amino acids as reported in the literature. Forexample, the epitopes of polyhistidine (His-tag), influenza aggregateHA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP),T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein(HSV-tag), E-tag (epitope on the monoclonal phage), and such, andmonoclonal antibodies to recognize these epitopes can be utilized as theepitope-antibody system for screening proteins binding to the protein ofthe present invention (Exp. Med. 13:85-90, 1995).

In immunoprecipitation, immune complexes are formed by adding theseantibodies to cell lysate prepared using suitable detergents. Thisimmune complex consists of a protein of the present invention, a proteincapable of binding to the protein, and an antibody. Theimmunoprecipitation can also be conducted using an antibody against aprotein of the present invention besides antibodies against the aboveepitopes. An antibody against the protein of the present invention canbe prepared, for example, by inserting a gene encoding a protein of thepresent invention into an appropriate E. coli expression vector toexpress the gene in E. coli, purifying the expressed protein, andimmunizing rabbits, mice, rats, goats, chickens, and such, with thepurified protein. The antibody can also be prepared by immunizing theabove-described animals with partial peptides of the protein of thepresent invention.

Immune complexes can be precipitated using, for example, Protein ASepharose or Protein G Sepharose in case where the antibody is a mouseIgG antibody. In addition, in the case where the protein of the presentinvention is prepared as a fusion protein with the epitope of, forexample, GST, and such, the immune complex can be formed using asubstance that specifically binds to these epitopes, such asglutathione-Sepharose 4B, and such, giving the same result as in thecase where the antibody for the protein of the present invention isused.

Immunoprecipitation, in general, may be performed following, oraccording to, for example, the method described in the literature(Harlow, E. and Lane, D.: Antibodies pp. 511-552, Cold Spring HarborLaboratory publications, New York, 1988).

SDS-PAGE is generally used for the analysis of immunoprecipitatedproteins, and bound proteins can be analyzed based on the molecularweight of proteins using a gel of an appropriate concentration. In thiscase, although proteins bound to a protein of the present invention, ingeneral, are hardly detectable by the usual protein staining methods,such as Coomassie staining and silver staining, the detectionsensitivity can be improved by culturing cells in a culture mediumcontaining radio isotope-labeled ³⁵S-methionine and ³⁵S-cystein to labelproteins inside the cells, and detecting the labeled proteins. Once themolecular weight of the protein is determined, the desired protein canbe purified directly from SDS-polyacrylamide gel and sequenced.

In addition, isolation of proteins binding to a protein of the presentinvention can be also performed using, for example, the West-Westernblotting analysis (Skolnik et al., Cell 65:83-90, 1991). Specifically, acDNA library is constructed from cells, tissues, and organs in whichprotein binding to the protein of this invention is expected to beexpressed by using phage vectors (such as, λgt11 and ZAP), proteinsexpressed on LB-agarose are fixed on a filter, which is then reactedwith a purified and labeled protein of the present invention, andplaques expressing proteins binding to the protein of the presentinvention are detected by the label. Methods for labeling a protein ofthe invention include methods utilizing the binding of biotin andavidin, methods utilizing antibodies specifically binding to the proteinof the present invention, or peptides or polypeptides (for example, GST,etc.) fused with the protein of the present invention, methods utilizingradioisotope and fluorescence, and such.

Further, another embodiment of the screening method of the presentinvention is exemplified by a method utilizing the two-hybrid systemusing cells (Fields et al., Trends Genet. 10:286-292, 1994; Dalton etal., Cell 68:597-612, 1992; “MATCHMAKER Two-Hybrid System”, “MammalianMATCHMAKER Two-Hybrid Assay Kit”, “MATCHMAKER One-Hybrid System” (allfrom Clontech); “HybriZAP Two-Hybrid Vector System” (Stratagene)). Inthe two-hybrid system, a protein of this invention may be fused to theDNA binding domain of SRF or GAL4, and expressed in yeast cells. A cDNAlibrary is constructed from cells predicted to express proteins thatbind to the protein of this invention, wherein the cDNA library isconstructed in such a way that the proteins are expressed as fusionproteins with transcription activation regions of VP16 or GAL4. The cDNAlibrary is transfected into the aforementioned yeast cells, and thenpositive clones are detected so as to isolate the cDNA-derived from thelibrary (i.e., expression of a protein that binds to the protein of theinvention in yeast cell leads to the binding of the two proteins, andresults in the activation of the reporter gene, which allows for thedetection positive clones). The protein encoded by the isolated cDNA maybe obtained by introducing the cDNA into E. coli and expressing ittherein. Thus, it is possible to prepare proteins that bind to a proteinof this invention and genes encoding them. The reporter gene to be usedin the two-hybrid system may be any suitable gene, such as HIS3 gene,Ade2 gene, LacZ gene, CAT gene, luciferase gene, or plasminogenactivator inhibitor type1 (PAI-1) gene, but is not limited thereto.

Screening for compounds which bind to a protein of the present inventioncan be also carried out using affinity chromatography. For example, aprotein of the invention is immobilized on a carrier in the affinitychromatography column, to which a test sample, which is expected toexpress a protein binding to the protein of the invention, is applied.Samples may be, for example, cell extracts, cell lysates, etc. Afterloading the test sample, the column is washed, and proteins which bindsto the protein of the invention can be obtained.

The obtained protein may be analyzed for its amino acid sequence tosynthesize oligonucleotide probes, which may then be used to screen acDNA library to obtain a DNA encoding the protein.

A biosensor that utilizes surface plasmon resonance phenomenon may beused to detect or measure the bound compound. Such biosensors (forexample, BIAcore (Pharmacia)) may enable the observation of theinteraction at real-time using a small amount of protein without theneed for labeling. Thus, it is possible to assess the interactionbetween the protein of the invention and test compounds using biosensorssuch as BIAcore.

Moreover, compounds that bind to a protein of the invention (includingagonists and antagonists), which compounds are not always proteins, maybe isolated using a variety of methods known to one skilled in the art.For instance, the protein of the invention may be fixed and exposed tosynthetic compounds, a bank of natural substances, or a random phagepeptide display library to screen for molecules that bind to theprotein. Alternatively, high-throughput screening using combinatorialchemistry techniques may be performed (Wrighton et al., Science273:458-64, 1996; Verdine, Nature 384:11-13, 1996; Hogan, Jr., Nature384:17-9, 1996).

Screening of a ligand that binds to a protein of the invention may beperformed as follows. The extracellular domain of a protein of theinvention is fused to the intracellular domain including thetransmembrane domain of a hemopoietin receptor protein that has a knownsignal transducing ability to prepare a chimeric receptor. The chimericreceptor may be expressed on the cell surface of a suitable cell line,preferably a cell line that can survive and proliferate only in thepresence of a suitable growth proliferative factor (growthfactor-dependent cell line). Then, the cell line may be cultured inmedium supplemented with a sample material in which a variety of growthfactors, cytokines, or hemopoietic factors might be expressed. Accordingto this method, the growth factor-dependent cell line can only surviveand proliferate when the sample contains an appropriate ligand thatspecifically binds to the extracellular domain of the protein of theinvention. The known hemopoietin receptors, such as the thrombopoietinreceptor, erythropoietin receptor, G-CSF receptor, gp 130, and so on maybe used. The partner for constructing a chimeric receptor for thescreening system of the invention is not limited to the above receptorsso long as its intracellular domain provides a structure necessary forthe signal transduction activity. The growth factor-dependent cell linemay be, for example, IL-3-dependent cell lines such as BaF3 or FDC-P1.

In a rare case, the ligand that specifically binds to a protein of theinvention may not be a soluble protein but a membrane-bound protein. Inthis case, screening can be done using a protein comprising only theextracellular domain of the protein of the invention, or a fusionprotein in which the extracellular domain is attached to a part of othersoluble proteins. Such proteins are labeled before they are used formeasuring the binding with the cells that are expected to express theligand. The former protein, comprising only the extracellular domain,may be a soluble receptor protein artificially constructed throughintroducing a stop codon into the N-terminal side of the transmembranedomain, or a soluble protein such as NR12-1. The latter fusion proteinmay be a protein in which the Fc region of immunoglobulin, or FLAGpeptide is attached to the C-terminus of the extracellular domain. Theselabeled soluble proteins can also be useful in detection by theabove-described West-western blotting method.

A chimeric protein of the extracellular domain of a protein of thisinvention and the Fc region of an antibody (such as human IgG antibody)may be purified using Protein A column, etc. Such an antibody-likechimeric protein retains its ligand binding activity. Thus, the proteinmay be appropriately labeled with an isotope and so on, and used for thescreening of a ligand (Suda et al., Cell 175:1169-1178, 1993). Somecytokines, such as molecules of the TNF family, primarily exist in amembrane-bound form, so such ligands may be isolated by exposing theantibody-like chimeric protein to a variety of cells and selecting cellsbased on the binding activity to the protein. Alternatively, ligands maybe isolated according to the same method by using cells to which a cDNAlibrary is introduced. Furthermore, the antibody-like chimeric proteinmay also be used as an antagonist.

The compound isolated by the above screening may be a candidate fordrugs that activate or inhibit the activity of a protein of thisinvention. It is possible to apply such compounds for the treatment ofthe disease arising from aberrant expression or functional disorder of aprotein of the present invention. The compound obtained using thescreening method of the invention includes compounds resulting from themodification of the compound having the activity to bind to the proteinof the invention by adding, deleting, and/or replacing a part of thestructure.

When using the isolated compound or a protein of the present invention(decoy type (soluble form)) as a pharmaceutical for humans and othermammals, for example, mice, rats, guinea pigs, rabbits, chicken, cats,dogs, sheep, pigs, bovines, monkeys, sacred baboons, chimpanzees, theisolated compound can be directly administered or can be formulated intoa dosage form using known pharmaceutical preparation methods. Forexample, according to the need, the pharmaceuticals can be taken orally,as sugar-coated tablets, capsules, elixirs and microcapsules, orparenterally, in the form of injections of sterile solutions,suspensions with water, or any other pharmaceutically acceptable liquid.For example, the compounds can be mixed with a pharmacologicallyacceptable carrier or medium, specifically, sterilized water,physiological saline, plant-oil, emulsifiers, suspending agent,surfactants, stabilizers, flavoring agents, excipients, vehicles,preservatives and binders, in a unit dosage form required for generallyaccepted drug implementation. The amount of active ingredient in thesepreparations makes a suitable dosage acquirable within the indicatedrange.

Examples of additives which can be mixed to tablets and capsulesinclude: binders, such as gelatin, corn starch, tragacanth gum and gumacacia; excipients, such as crystalline cellulose; swelling agents, suchas corn starch, gelatin and alginic acid; lubricants, such as magnesiumstearate; and sweeteners, such as sucrose, lactose or saccharin;flavoring agents, such as peppermint, Gaultheria adenothrix oil andcherry. When the unit dosage form is a capsule, a liquid carrier, suchas oil, can also be included in the above ingredients. Sterilecomposites for injections can be formulated following normal drugimplementations, using vehicles such as distilled water used forinjections.

For example, physiological saline, glucose, and other isotonic liquids,including adjuvants, such as D-sorbitol, D-mannose, D-mannitol, andsodium chloride, can be used as aqueous solutions for injections. Thesecan be used in conjunction with suitable solubilizers, such as alcohol,specifically ethanol, polyalcohols such as propylene glycol andpolyethylene glycol, and non-ionic surfactants, such as Polysorbate 80(TM) and HCO-50.

Sesame oil or soy-bean oil can be used as an oleaginous liquid and maybe used in conjunction with benzyl benzoate or benzyl alcohol assolubilizers; may be formulated with a buffer such as phosphate bufferand sodium acetate buffer; and may be used in conjunction with apain-killer such as procaine hydrochloride, a stabilizer such as benzylalcohol and phenol, and an anti-oxidant. The prepared injection isfilled into a suitable ampule.

Methods well known to those skilled in the art may be used to administerthe pharmaceutical compound to patients, for example as intraarterial,intravenous, percutaneous injections and also as intranasal,transbronchial, intramuscular or oral administrations. The dosage variesaccording to the body-weight and age of the patient, the administrationmethod, and such, but one skilled in the art can suitably select them.If said compound is encodable by a DNA, said DNA can be inserted into avector for gene therapy to perform the therapy. The dosage and method ofadministration vary according to the body-weight, age, and symptoms of apatient, but one skilled in the art can select them suitably.

For example, the dosage of the protein of this invention (decoy form(soluble form)) may vary depending on the subject of administration,target organ, symptom, and method for administration. However, it may beinjected to a normal adult (body weight, 60 kg) at a dose of about 100μg to 10-20 mg per day.

For example, although there are some differences according to thesymptoms, the dose of a compound that binds with a protein of thepresent invention, or a compound that inhibits the activity of a proteinof this invention is typically about 0.1 mg to about 100 mg per day,preferably about 1.0 mg to about 50 mg per day, and more preferablyabout 1.0 mg to about 20 mg per day, when administered orally to anormal adult (weight 60 kg).

When the protein is administered parenterally, in the form of aninjection to a normal adult (weight 60 kg), although there are somedifferences according to the patient, target organ, symptoms and methodof administration, it is convenient to intravenously inject a dose ofabout 0.01 mg to about 30 mg per day, preferably about 0.1 to about 20mg per day and more preferably about 0.1 to about 10 mg per day. Also,in the case of other animals, it is possible to administer an amountconverted to 60 kg of body-weight or surface area.

All publications and patents cited herein are incorporated by referencein their entirety.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the partial nucleotide sequence of AL109843 identified inthe htgs database (SEQ ID NO:23). The deduced amino acid sequence isshown under the predicted exon sequence (SEQ ID NO:24). The YR motifsequence and WS motif that were used as the target are boxed.

FIG. 2 shows partial amino acid sequences of NR12 found in the sequenceof AL109843 (from the top, SEQ ID NOs:25-29), and those of knownhemopoietin receptors having homology thereto. Identical amino acidsequences are boxed and similar amino acid sequences are shadowed. Gapspaces are underlined. Known hemopoietin receptors are, from top, humangp130 (SEQ ID NO:30), human NR9 (SEQ ID NO:31), human prolactin receptor(SEQ ID NO:32), human IL-7 receptor (SEQ ID NO:33), and human LIFreceptor (SEQ ID NO:34).

FIG. 3 shows a photograph demonstrating the results of PCR analysis,showing expressed products amplified by 5′-RACE and 3′-RACE using theoligonucleotide primers designed against the predicted WS exon withinthe AL109843 sequence. Specific products by PCR are shown with arrows.

FIG. 4 shows the nucleotide sequence of the full-length NR12.1 cDNA thatwas obtained by combining the 5′-RACE and 3′-RACE products (SEQ IDNO:1). The deduced amino acid sequence encoded by NR12.1 is also shown(SEQ ID NO:2). The amino acid sequence predicted to be the secretionsignal is underlined. Conserved cysteine residues, and the amino acidsequences of YR motif and WS motif are boxed.

FIG. 5 shows the nucleotide sequence of the full-length NR12.2 cDNA thatwas obtained by combining the 5′-RACE and 3′-RACE products (SEQ IDNO:3). The amino acid sequence encoded by NR12.2 is also shown (SEQ IDNO:4). The predicted secretion signal sequence is underlined. Thepredicted transmembrane region is shadowed. Conserved cysteine residuesin the extracellular region, and amino acid sequences of YR motif and WSmotif are boxed.

FIG. 6 shows the nucleotide sequence of full-length NR12.3 cDNA that wasobtained by combining the 5′-RACE and 3′-RACE products (SEQ ID NO:5).The amino acid sequence encoded by NR12.3 is also shown (SEQ ID NO:6).The predicted secretion signal is underlined. Conserved cysteineresidues, and the amino acid sequences of YR motif and WS motif areboxed.

FIG. 7 is a continuation of FIG. 6.

FIG. 8 shows photographs demonstrating the results of RT-PCR analysis ofthe genetic-expression distribution of the NR12 in human organs. Thearrow indicates the size of the specific PCR amplification product ofNR12.

FIG. 9 shows a photograph demonstrating the results of quantification ofthe NR12 gene expression in human organs by Southern blotting. The arrowindicates the size of the specific signal of detected NR12.

FIG. 10 is a schematic illustration of the structure of the NR12 fusionprotein to be expressed from the expression vector construct in themammalian cell.

FIG. 11 shows the nucleotide sequence of full-length NR12.4 cDNA thatwas obtained by combining the 5′-RACE and 3′-RACE products (SEQ IDNO:7). The amino acid sequence encoded by NR12.4 is also shown (SEQ IDNO:8). The predicted secretion signal is underlined. Conserved cysteineresidue, and amino acid sequences of YR motif and WS motif are boxed.

FIG. 12 is a continuation of FIG. 11.

FIG. 13 shows the nucleotide sequence of full-length NR12.5 cDNA (SEQ IDNO:9). The amino acid sequence encoded by NR12.5 is also shown (SEQ IDNO:10). The predicted secretion signal is underlined. The predictedtransmembrane sequence is shaded. Conserved cysteine residue in theextracellular region, and amino acid sequences of YR motif and WS motifare boxed.

FIG. 14 is a continuation of FIG. 13.

DETAILED DESCRIPTION

The present invention will be explained below with reference toexamples, but it is not construed as being limited thereto.

Example 1 Isolation of NR12 Gene (1) Primary Screening by TblastN Search

Although sequencing of human genome is promoted extensively by humangenome projects of institutes, the proportion of completely finishedsequences to the whole human genome has not reached even 10%. However,information provided by above projects until today is counted as a goodmeans for searching target genes, determining nucleotide sequences, andmapping genes. The informational basis of the above sequences consistsof large information provided by the assembly of bacterial artificialchromosome (BAC) and yeast artificial chromosome (YAC), which aims toform a complete database in the future. The present inventors identifieda human gene encoding a part of a novel hemopoietin receptor proteinfrom a BAC clone sequence in one of public databases, “High ThroughputGenomic Sequence (htgs)” of GenBank.

As mentioned above, the present inventors found motif sequencesconserved in the hemopoietin receptor family, namely(Tyr/His)-Xaa-(Hydrophobic/Ala)-(Gln/Arg)-Hydrophobic-Arg motif (YRmotif) in the extracellular region, and Trp-Ser-Xaa-Trp-Ser (SEQ IDNO:21) motif (WS motif) located around the C-terminus. However, it isextremely difficult to design oligonucleotide probe that includes bothmotif sequences comprehensively. Therefore, the inventors conducted insilico database search using partial amino acid sequences from thefragment of known hemopoietin receptor proteins including both motifs asthe query. Fragmentation of partial amino acid sequences that may beused as a query was examined using the human receptors shown in table 1as the sequence of known hemopoietin receptors. According to the genomicstructure of the known hemopoietin receptor sequences, the exonsencoding these YR motif and WS motif were about 50 to 70 amino acidslong, and the exon proximal to it to the N-terminus (PP exon) was alsoabout 50 to 70 amino acids long. Thus, a sequence containing both exonsconsisting of about 120 amino acids were cut to prepare a query sequencefor convenience' sake. Although the length of the partial amino acidsequence used as the query sequence varied depending on each knownhemopoietin receptor, the feature of the structure was conserved. Asequence that ranges from one or more proline residues located near theinitiation site in the PP exon to the amino acid residue located about10 amino acids to the C-terminus of the WS motif termination in the WSexon was extracted as the query sequences from all known hematopoietinreceptor sequences.

The known hemopoietin receptors used as query sequences for the databasesearch is shown in the table. The amino acid residues conserved amongmotif sequences are shown in bold with underline.

TABLE 1 Genbank Human Accession YR-motif WS-motif Receptors # SequenceSequence LIF-R NM_002310 Y TFRI R WS K WS (SEQ ID NO:35) (SEQ ID NO:57)gp130 NM_002184 Y VFRI R WS D WS (SEQ ID NO:36) (SEQ ID NO:58) IL-12Rβ1NP_005526 QEFQL R WS K WS (SEQ ID NO:37) (SEQ ID NO:57) IL-12Rβ2NM_001559 Y EFQIS WS D WS (SEQ ID NO:38) (SEQ ID NO:58) G-CSFR NM_000760Y TLQI R WS D WS (SEQ ID NO:39) (SEQ ID NO:58) EPO-R M34986 Y TFAV R WSA WS (SEQ ID NO:40) (SEQ ID NO:59) TPO-R M90103 Y RLQL R WS S WS (SEQ IDNO:41) (SEQ ID NO:60) Leptin-R U50748 Y AVQV R WS N WS (SEQ ID NO:42)(SEQ ID NO:61) IL-3Rα M74782 Y TVQI R L S A WS (SEQ ID NO:43) (SEQ IDNO:62) IL-4R NM_000418 Y RARV R WS E WS (SEQ ID NO:44) (SEQ ID NO:63)IL-5Rα M96651 Y DVQV R WS E WS (SEQ ID NO:45) (SEQ ID NO:63) IL-6RNM_000565 HVVQL R WS E WS (SEQ ID NO:46) (SEQ ID NO:63) IL-7R NM_002185Y EIKV R WS E WS (SEQ ID NO:47) (SEQ ID NO:63) IL-11Rα U32324 HAVRVS WST WS (SEQ ID NO:48) (SEQ ID NO:64) IL-13Rα NM_001560 NTVRI R WS N WS(SEQ ID NO:49) (SEQ ID NO:61) IL-2Rβ A28052 Y EFQV R WS P WS (SEQ IDNO:50) (SEQ ID NO:65) IL-2Rγ NM_000206 Y TFRV R WS E WS (SEQ ID NO:51)(SEQ ID NO:63) GM-CSFR M64445 HSVKI R WS S WS (SEQ ID NO:52) (SEQ IDNO:60) CNTF-R NM_001842 Y IIQVA WS D WS (SEQ ID NO:53) (SEQ ID NO:58)PRL-R NM_000949 Y LVQV R WS A WS (SEQ ID NO:54) (SEQ ID NO:59)NR6(CRLF1) NM_004750 Y FVQV R WS E WS (SEQ ID NO:55) (SEQ ID NO:63)NR9(CREME9) AF120151 Y QERVC WS P WS (SEQ ID NO:56) (SEQ ID NO:65)

The above queries were used to search on the htgs database in GenBankusing TblastN (Advanced TblastN 2.0.9) program. The default values(Expect=100, Descriptions=250, and Alignments=250) were used asparameters for the search. As a result, the search resulted in manyfalse positive clones, and those clones which both of the YR motif andWS motif were not encoded in the same reading frame, or that contained astop codon between the two motifs were excluded. Also those clonescontaining only the YR motif but not the WS motif were also excluded,because, as mentioned above, the YR motif is not a completelyestablished consensus sequence. Therefore, the conservation of the WSmotif was considered predominant. As a result of the above selection,positive clones of primary search shown in table 2 were chosen fromabout 1000 pseudo-positive clones obtained by the TblastN search.

Positive clones obtained by the primary search against htgs databasehaving the target motif sequence with high probability were selected,and are shown in the table. Conserved amino acid residues are shown inbold with underline in the motif sequences.

TABLE 2 GenBank Motif Accession # Sequence Note AC008048 WS P WS IL-2Rbeta (SEQ ID NO:65) AC007174 WS E WS IL-5R (SEQ ID NO:63) AL031406 WS TWS CH.22 (SEQ ID NO:64) AC003656 WS G WS CH.21 (SEQ ID NO:66) AC008663WS K WS CH.5 (SEQ ID NO:57) AC008614 WS G WS CH.5 (SEQ ID NO:66)AC008532 WS G WS CH.19 (SEQ ID NO:66) AC009267 WS T WS CH.18 (SEQ IDNO:64) AC007596 WS S WS CH.16 (SEQ ID NO:60) AC007227 W GE WS CH.16 (SEQID NO:67) AL031123 WS D W A CH.6 (SEQ ID NO:68) AC005911 W GE WS CH.12(SEQ ID NO:67) AL096870 WS N W K CH.14 (SEQ ID NO:69) Z97201 WS N W KCH.12 (SEQ ID NO:69) AC007902 WS G WS CH.18 (SEQ ID NO:66) AC008536 WS MWS CH.5 (SEQ ID NO:70) AC006176 WS G WS CH.10 (SEQ ID NO:66) AC004846 WSQ WS none (SEQ ID NO:71) AL109843 W QP WS CH.1 (NR12) (SEQ ID NO:72)AC003656 WS E W G CH.21 (SEQ ID NO:73) AC005143 T S G WS CH.15 (SEQ IDNO:74) AL109743 WS G WS CH.1 (SEQ ID NO:66) AC008403 WS A WS CH.19 (SEQID NO:59) AL032818 WS G WS CH.22 (SEQ ID NO:66) Z93017 WS G WS CH.6 (SEQID NO:66) AC009456 WS R WS CH.18 (SEQ ID NO:75) AC008427 WS EG S CH.5(SEQ ID NO:76) AL096791 WS Q WS CH.X (SEQ ID NO:71)

(2) Secondary Screening by BlastX Search

First, nucleotide sequences around the sequence which were positive tothe query sequence in the primary search were cut from each of the 28positive clones of TblastN primary search shown in table 2. Using thesesequences as the query, the nr database in GenBank was searched againusing the BlastX (Advanced BlastX 2.0.9) program. The query sequenceconsisted of a nucleotide sequence of 240 bp in total, which containsthe sequence approximately 200 bp upstream of the sequence that mayencode the WS motif, for convenience sake. Because, as mentioned above,the exon encoding the WS motif was as short as approximately 50 to 70amino acids in the genome structure of known hemopoietin receptors, theprepared query sequence of 240 bp long is expected to cover the exonsufficiently. The value of “Expect=100, Descriptions=100,Alignments=100, Filter=default” was used for the BlastX search. It wasexpected that positive clones showing at least homology with multipledifferent known hemopoietin receptors would be selected as positiveclones of secondary search encoding hematopoietin receptor familymembers from the positive clones of the secondary search according tothe search.

As a result of the above two-step Blast search, three clones (AC008048,AC007174, and AL109843) among the human genome clones shown in table 2were successfully identified as positive clones of the secondary search.However, AC008048 and AC007174 were revealed to be genome sequences thatencode the human IL-2 receptor beta strand and human IL-5 receptor,respectively. AL109843 alone was inferred to encode the target novelhemopoietin receptor. Therefore, this clone was named NR12, and wasdetermined to isolate the full-length cDNA.

AL109843 is a genome draft sequence derived from human chromosome 1submitted to htgs database at 16 Aug. 1999, and has a length of 149104bp. However, nucleotide sequences at 10 positions, approximately 8000 bpin total, remains undetermined at this time. The existence of a WS exoncould be predicted in the sequence of AL109843 which were positive inthe TblastN primary search as shown in FIG. 1. The YR motif, [YVFQVR;SEQ ID NO:77] sequence, and WS motif, [WQPWS; SEQ ID NO:72] sequence,was recognized in the sequence. The comparison of the amino acidsequence of NR12 to that of the known hematopoietin receptor, which weredetected to have homology in BlastX secondary search, are shown in FIG.2. Based on the above result, specific oligonucleotide primers weredesigned on the exon sequence that were predicted in the AL109843sequence, and these primers were used in the 5′-RACE method and the3′-RACE method described later on.

(3) Design of Oligonucleotide Primers

As described above, exon sites were predicted on AL109843 sequences, andthese were used to design the following oligonucleotide primers specificfor NR12. Three sense primers (NR12-S1, NR12-S2, and NR12-S3; orienteddownstream) and three antisense primers (NR12-A1, NR12-A2, and NR12-A3;oriented upstream) were synthesized using the ABI 394 DNA/RNAsynthesizer under a condition to attach a trityl group to the5′-terminus. Then, the products were purified using an OPC column (ABI#400771) to obtain full-length primers.

NR12-S1; (SEQ ID NO:11) 5′- GCA ACA GTC AGA ATT CTA CTT GGA GCC-3′NR12-S2; (SEQ ID NO:12) 5′- CAT TAA GTA CGT ATT TCA AGT GAG ATG TC -3′NR12-S3; (SEQ ID NO:13) 5′- GGT ACT GGC AGC CTT GGA GTT CAC TG -3′NR12-A1; (SEQ ID NO:14) 5′- CAG TGA ACT CCA AGG CTG CCA GTA CC -3′NR12-A2; (SEQ ID NO:15) 5′- GAC ATC TCA CTT GAA ATA CGT ACT TAA TG -3′NR12-A3; (SEQ ID NO:16) 5′- GGC TCC AAG TAG AAT TCT GAC TGT TGC -3′

Above oligonucleotide primers, NR12-S1 and NR12-A3, NR12-S2 and NR12-A2,and NR12-S3 and NR12-A1 were designed to have completely complementarysequence to each other.

(4) Cloning of N-Terminal cDNA by 5′-RACE Method

In order to isolate full-length cDNA of NR12, 5′-RACE PCR was performedusing NR12-A1 of (3) for primary PCR, and NR12-A2 of (3) for secondaryPCR, respectively. PCR experiment was performed using Human Fetal LiverMarathon-Ready cDNA Library (Clontech #7403-1) as the template, andAdvantage cDNA Polymerase Mix (Clontech #8417-1) on the thermal cycler(Perkin Elmer Gene Amp PCR System 2400). Under the following conditions,as a result, PCR products of two different sizes were obtained as shownin FIG. 3.

Condition of the primary PCR was as follows: 94° C. for 4 min, 5 cyclesof “94° C. for 20 sec, 72° C. for 90 sec”, 5 cycles of “94° C. for 20sec, 70° C. for 90 sec”, 28 cycles of “94° C. for 20 sec, 68° C. for 90sec”, 72° C. for 3 min, and termination at 4° C.

Condition of the secondary PCR was as follows: 94° C. for 4 min, 5cycles of “94° C. for 20 sec, 70° C. for 90 sec”, 25 cycles of “94° C.for 20 sec, 68° C. for 90 sec”, 72° C. for 3 min, and termination at 4°C.

Two amplification products were obtained by the PCR and both of themwere subcloned into pGEM-T Easy vector (Promega #A1360), and thenucleotide sequences were determined. The transformation of the PCRproduct into the pGEM-T Easy vector was performed using T4 DNA ligase(Promega #1360) in a reaction at 4° C. of 12 hours. Recombinants of thePCR products and pGEM-T Easy vector were obtained by the transformationof E. coli DH5α strain (Toyobo #DNA-903). Recombinants were selected byusing Insert Check Ready Blue (Toyobo #PIK-201). The nucleotidesequences were determined using the BigDye Terminator Cycle SequencingReady Reaction Kit (ABI/Perkin Elmer #4303154) and by analyzing with theABI PRISM 377 DNA Sequencer. Nucleotide sequences of the whole insertfragment of 10 independent clones were determined. As a result, theywere divided into two groups, one consisting of 4 clones with a size of1.3 kb, and the other consisting of 6 clones with a size of 1.0 kb,based on the length of the base pairs and the differences in sequence.However, the former 5′-RACE PCR products of 1.3 kb were revealed to benon-specific PCR amplification products. This sequence is derived fromthe minor band shown in FIG. 3. On the other hand, the latter 5′-RACEPCR products of 1.0 kb were recognized as partial nucleotide sequencesof NR12 that resulted from a correct PCR amplification reaction.

(5) Cloning of C-Terminal cDNA by 3′-RACE Method

To isolate the C-terminal sequence of a cDNA clone corresponding to thefull-length NR12.3′-RACE PCR was performed using NR12-S1 primer of (3)for the primary PCR, and NR12-A2 of (3) for secondary PCR, respectively.The PCR was performed under the same condition as in the 5′-RACE aboveexcept the Human Thymus Marathon-Ready cDNA Library (Clontech#7415-1)was used as the template. More specifically, Advantage cDNA PolymeraseMix and the Perkin Elmer Gene Amp PCR System 2400 thermalcycler was usedin the PCR experiment. Under the same PCR condition to those describedin (4), 3′-RACE amplification product showing an identical size of 750bp was obtained as shown in FIG. 3. The obtained PCR product wassubcloned into the pGEM-T Easy vector as above to determine thenucleotide sequence. The recombination of the PCR product into thepGEM-T Easy vector was performed using T4 DNA ligase in a reaction at 4°C. for 12 hours. The recombinant of the PCR product and pGEM-T Easyvector was obtained by transformation of E. coli DH5α strain, andselection of the recombinant was done using Insert Check Ready Blue asdescribed above. The nucleotide sequence was determined using the BigDyeTerminator Cycle Sequencing Ready Reaction Kit and the ABI PRISM 377 DNASequencer for analysis. The nucleotide sequences of the whole insertfragment from 2 independent clones of genetic recombinants revealed thatthe clones contain the C-terminal sequence of the full-length NR12 cDNAclone having a poly A sequence.

Then, the nucleotide sequence determined by the 3′-RACE-PCR and thosedetermined by 5′-RACE-PCR in (4) were combined to finally determine thewhole nucleotide sequence of the cDNA clone encoding the secretory formsoluble receptor-like protein named NR12.1. The determined nucleotidesequence of NR12.1 cDNA (SEQ ID NO:1) and the amino acid sequenceencoded by the sequence (SEQ ID NO:2) are shown in FIG. 4.

(6) Cloning of a C-Terminal Splicing Variant by 3′-RACE Method

Although the NR12.1 clone isolated above had sufficient feature of knownhemopoietin receptors according to the result of structural analysis, itdid not possess a transmembrane region. Therefore, it was inferred toencode a soluble receptor-like protein as above-mentioned. Further, thepresent inventors predicted the existence of splicing variants that havea transmembrane region especially in the C-terminal region of thetranscription product of the present gene, and tried to isolate NR12cDNA clones by successive 3′-RACE method.

Thus, 3′-RACE PCR was performed using the above-mentioned NR12-S2 primerof (3) for primary PCR, and NR12-S3 primer for secondary PCR. Under thesame PCR condition to those described in (4) for 5′-RACE method exceptusing Human Testis Marathon-Ready cDNA Library (Clontech#7414-1) as thetemplate. As a result, multiple 3′-RACE PCR products with differentsizes were obtained. All of the obtained PCR products were subclonedinto the pGEM-T Easy vector as described above to determine thenucleotide sequences. Nucleotide sequences of the whole insert fragmentsof 6 independent clones of genetic recombinants were determined. As aresult, one of these clones was found to be identical to NR12.1determined above. The other 5 clones were possible to encode the targettransmembrane protein having transmembrane regions. That is, the presentinventors were able to confirm the existence of splicing variants ofNR12 as expected. Furthermore, the 5 cDNA clones above showeddifferences in the C-terminal extracellular region due to alternativesplicing. Namely, two of these clones had only a short intracellularregion and were named NR12.2. On the other hand, the other 3 clones hada long intracellular region. These cDNA clones with a long ORF werenamed NR12.3, and were distinguished from the above sequence, NR12.2.

Then, the nucleotide sequence determined by the 3′-RACE PCR and thosefrom the 5′-RACE PCR products in (4) were combined to finally determinethe whole nucleotide sequence of the cDNA clone that encodes thetransmembrane receptor protein. The nucleotide sequence determined forNR12.2 cDNA (SEQ ID NO:3) and its amino acid sequence (SEQ ID NO:4) areshown in FIG. 5. The nucleotide sequence of NR12.3 cDNA (SEQ ID NO:5)and its amino acid sequence (SEQ ID NO:6) are shown in FIGS. 6 and 7.

The exon site sequence was predicted in above (2) from the splicingconsensus sequence in RNA transcription (Hames, B. D. and Glover, D. M.,Transcription and Splicing (Oxford, IRL Press), 1988, p131-206) and notby using program such as genome analysis software. According to thedetermination of the whole nucleotide sequence of isolated cDNA clones,it was revealed that the exon site predicted within the partial sequenceof AL109843 shown in FIG. 1 correspond completely to that observed inthe actual transcription of the NR12 gene. However, it was revealed thatonly the transcription product of NR12.1 cDNA clone was one whichelongates to the 3′-untranslated region read through the identicalsequence as that of the genome structure without splicing after thetermination of WS exon.

(7) Structural Feature of NR12 and Prediction of its Function

As a result of the determination of the whole nucleotide sequences ofNR12.1, NR12.2 and NR12.3, it was revealed that they are thetranscription products having structural variety in the C-terminus dueto alternative splicing. The NR12.1 may encode a secretory form solublehemopoietin receptor-like protein consisting of 337 amino acidsaccording to its primary structure, while the NR12.2 and NR12.3 mayencode transmembrane hemopoietin receptor proteins consisting of 428 and629 amino acids, respectively. The characteristics of each NR12 were asfollows.

First, it is predicted that the sequence from the 1^(st) Met to the23^(rd) Gly in the common extracellular domain of these clones is thetypical secretion signal sequence. Herein, the first Met is presumed tobe the translation initiation site because there exists an in-frametermination codon at the minus 32 position from the 1^(st) Met. Next, anIg-like region exists in the region from the 24^(th) Gly to the 124^(th)Pro residue. In addition, it is predicted that the region from the133^(rd) Cys to the 144^(th) Cys residue forms one of the loopstructures which is a ligand-binding site. Furthermore, the region fromthe 290^(th) Tyr to the 295^(th) Arg residue corresponds to the highlyconserved YR motif, and a typical WS motif is also found at residuesfrom the 304^(th) Trp to 308^(th) Ser.

Herein, the NR12.1 encodes 29 amino acids after the WS motif and thetranslation frame terminates at the next stop codon. Therefore, theNR12.1 encodes a soluble hemopoietin receptor protein that does not havea transmembrane domain. On the other hand, the 26 amino acids followingthe conserved motif above from the 352^(nd) Gly to the 377^(th) Asnresidue in NR12.2 and NR12.3 correspond to a typical transmembranedomain. The NR12.2 and NR12.3 encode identical amino acid sequences tothe 413^(th) Gln residue in the extracellular region. However,structural differences exist in the C-terminal region following the413^(th) Gln residue due to alternative splicing which connects them todifferent exons. Namely, NR12.2 encodes 428 amino acids and thetranslation frame is terminated at the next stop codon. Thus, it hasonly a short intracellular region consisting of 51 amino acids. On theother hand, NR12.3 encodes 629 amino acids and has an intracellularregion consisting of 252 amino acids. According to the structuralcharacteristics above, NR12 gene was recognized to possess sufficientcharacteristics as novel hemopoietin receptor proteins.

Example 2 Tissue Distribution Determination and Expression PatternAnalysis of NR12 Gene by RT-PCR

mRNA was detected using the RT-PCR method to analyze the expressiondistribution and the expression pattern of NR12.1 gene in differenthuman organs. NR12-PPD primer with the sequence below was synthesized asa sense primer (downstream orientation) for the RT-PCR analysis. NR12-A1primer synthesized in Example 1 (3) was used as the antisense primer(upstream orientation). The NR12-PPD primer was synthesized and purifiedas in Example 1 (3). It was expected that the common N-terminal regionin all splice variants, NR12.1, NR12.2 and NR12.3, are amplified anddetected using these primer sets (NR12-PPD and NR12-A1).

hNR12-PPD; (SEQ ID NO:17) 5′- CCG CCA GAT ATT CCT GAT GAA GTA ACC -3′

The templates used were Human Multiple Tissue cDNA (MTC) Panel I(Clontech #K1420-1), Human MTC Panel II (Clontech #K1421-1), HumanImmune System MTC Panel (Clontech #K1426-1), and Human Fetal MTC Panel(Clontech #K1425-1). PCR was performed using Advantage cDNA PolymeraseMix (Clontech #8417-1) on a thermal cycler (Perkin Elmer Gene Amp PCRSystem 2400). PCR was performed by following condition to amplify thetarget gene: 94° C. for 4 min, 5 cycles of “94° C. for 20 sec, 72° C.for 1 min”, 5 cycles of “94° C. for 20 sec, 70° C. for 1 min”, 25 cyclesof “94° C. for 20 sec, 68° C. for 1 min”, 72° C. for 3 min, andtermination at 4° C.

As shown in FIG. 8, strong expression of NR12 was observed in thehematopoietic cell line tissue and immune system cell line tissue suchas adult spleen, thymus, lymph node, bone marrow, and peripheralleukocyte. Expression was also detected in testis, liver, lung, kidney,pancreas, and gastrointestinal tract such as small intestine and colon.Moreover, NR12 gene expression was also observed in all analyzed mRNAderived from human fatal tissues. Performing PCR using human G3PDHprimers under the above condition and detecting the expression of thehousekeeping gene G3PDH, it was confirmed that the number of mRNA copiesamong the template mRNA had been normalized.

The size of the RT-PCR amplification product was 561 bp, which wasconsistent with the size calculated from the determined nucleotidesequence of NR12 cDNA. Thus, the product was considered to be theproduct of specific PCR amplification reaction. This was furtherconfirmed by Southern blotting as in the following, and the possibilitythat the product was a non-specific PCR amplification product wasdenied.

The analyses of expression distribution and expression pattern of NR12gene by RT-PCR revealed that the expression is restricted to specificorgans and tissues, and also that the amount of expression variesgreatly among organs. Taking all the result of NR12 gene expressiondistribution together, the fact that especially strong expression wasdetected in tissue considered mainly to include immunocyte tissues andhematopoietic cells suggest strongly the possibility that NR12 functionsas a novel hemopoietin receptor. Furthermore, the fact that theexpression of NR12 was also observed in other tissues suggests that NR12can regulate various physiological functions in vivo not only those inthe immune system and hematopoietic system.

Moreover, existence of splicing variants was recognized. This stronglysuggests that transcriptional regulation of the NR12 gene expression isstrictly controlled by the transcriptional regulation determiningfunctional specificity, transcriptional induction by exogenousstimulating factor, and regulation of alternative splicing in specificcell types.

Example 3 Verification of the Specificity of RT-PCR Product by SouthernBlotting

In order to verify the specificity of amplification, the RT-PCRamplified target gene product in Example 2 was subjected to Southernblotting using NR12 specific cDNA fragment as a probe. At the same time,the amount of RT-PCR product was quantitatively detected by the strengthof labeled signal to assess relative gene expression levels amongdifferent human organs. The RT-PCR product was electrophoresed on anagarose gel, blotted onto a charged nylon membrane, Hybond N (+)(Amersham, cat #RPN303B), and was subjected to hybridization. The5′-RACE PCR product cDNA fragment corresponding to the N-terminus of theNR12 obtained in Example 1 (4) was used as a probe specific to NR12.Probes were prepared using the Mega Prime Kit (Amersham, cat #RPN1607),and labeled with radioisotope, [α-³²P] dCTP (Amersham, cat #AA0005).Hybridization was performed using Express Hyb Hybridization Solution(Clontech #8015-2), and after the prehybridization at 68° C. for 30 min,heat-denatured labeled probe was added to conduct hybridization at 68°C. for 120 min. After subsequent wash in (1) 1×SSC/0.1% SDS at roomtemperature for 5 min; (2) 1×SSC/0.1% SDS at 50° C. for 30 min; and (3)0.1×SSC/0.1% SDS at 50° C. for 30 min, the membrane was exposed toImaging Plate (FUJI #BAS-III), and NR12 specific signal was detected bythe Image Analyzer (FUJIX, BAS-2000 II).

As shown in FIG. 9, all the amplified PCR products by the RT-PCR abovewere verified as specific amplification products. Furthermore, theresult of quantification of relative expression level among each tissuealso supported above-mentioned assessment. The detection method fortarget gene expression using RT-PCR and Southern blotting in combinationis known to have extremely high sensitivity as compared to other methodsfor expression analysis. Nevertheless, NR12 gene expression was notdetected in adult heart, skeletal muscle, adult brain, prostate, ovary,or placenta at all.

Example 4 Northern Blot Analysis of NR12 Gene Expression

Northern blot analysis of NR12 gene expression was performed to examinethe expression pattern of NR12 gene in human organs and human cancercell lines, and to determine the size of NR12 transcripts. HumanMultiple Tissue Northern (MTN) Blot (Clontech #7760-1), Human MTN BlotII (Clontech #7759-1), Human MTN Blot III (Clontech #7767-1), and HumanCancer Cell Line MTN Blot (Clontech #7757-1) were used.

The cDNA fragment obtained by 5′-RACE in Example 1 (4) was used as theprobe. The probe was prepared using Mega Prime Kit and radio-labeledwith [α-³²P]dCTP as in Example 3. Hybridization was performed usingExpress Hybridization Solution, and after prehybridization at 65° C. for30 min heat-denatured labeled probe was added to conduct hybridizationat 65° C. for 16 hr. After subsequent wash in (1) 1×SSC/0.1% SDS at roomtemperature for 5 min; (2) 1×SSC/0.1% SDS at 48° C. for 30 min; and (3)0.5×SSC/0.1% SDS at 48° C. for 30 min, the membrane was exposed to anImaging Plate as above, and an attempt to detect NR12 specific signalwas made using an Image Analyzer.

The method failed to detect any signal in any of the examined humanorgans. This could be because Northern blotting has a significant lowersensitivity than RT-PCR and thus failed to detect mRNA with lowexpression level.

Example 5 Construction of an NR12 Ligand Screening System Using GrowthFactor-Dependent Cell Lines

Ligands that bind specifically to the protein of this invention can bescreened by the following step: (1) preparing a chimeric receptor byligating the extracellular domain of the protein of this invention withthe intracellular domain containing the transmembrane domain of ahemopoietin receptor protein comprising a known signal transductionability; (2) expressing this chimeric receptor on the cell surface of asuitable cell line, preferably, a cell line that can survive andproliferate only under the presence of a suitable factor (a growthfactor-dependent cell line); and (3) culturing the cell line by adding amaterial that is expected to contain various growth factors, cytokines,or hemopoietic factors. This method utilizes the fact that theabove-mentioned growth factor-dependent cell line only survives andproliferates when a ligand specifically binding to the extracellulardomain of the protein of the invention exists within the test materialand is killed rapidly without the existence of the growth factor. Knownhemopoietic receptors are, for example, the thrombopoietin receptor,erythropoietin receptor, G-CSF receptor, gp130, etc. However, thepartner of the chimeric receptor used in the screening of the inventionis not limited to these known hemopoietic receptors, and any receptormay be used so long as it contains the structure necessary for thesignal transducing activity in the cytoplasmic domain. IL-3-dependentcell lines, such as Ba/F3 and FDC-P1, can be exemplified as growthfactor-dependent cell lines.

First, the cDNA sequence encoding the extracellular region of NR12 (theamino acid sequence from the 1^(st) Met to the 319^(th) Gly) wasamplified by PCR, and this DNA fragment was bound in frame to the DNAfragments encoding the transmembrane region and the intracellular regionof a known hemopoietin receptor to prepare a fusion sequence encoding achimeric receptor. The TPO receptor (Human MPL-P) was selected from thecandidates described above as the known partner hemopoietin receptor.The constructed chimeric receptor sequence above was inserted into theplasmid vector, pME18S/neo, which can be expressed in mammalian cells. Aschematic diagram of the structure of the constructed pME18S/NR12-TPORchimeric receptor is shown in FIG. 10. The chimeric receptor-expressingvector was introduced into the growth factor-dependent cell line Ba/F3,and was forced to express. Then, stable gene-introduced cells wereselected. The selection can be done by utilizing the fact that theexpression vector contains a drug (neomycin) resistant gene, and thus,only gene-introduced cells that obtained drug tolerance can beproliferated in the culture containing the drug. Novel hematopoietin maybe screened by constructing a screening system that utilizes the abilityof the chimeric receptor-expressing cell lines to survive andproliferate only under the existence of a ligand functionally bindingspecifically to the NR12. In this case, the culture of the chimericreceptor-expressing cell line is conducted in medium supplemented with amaterial expected to include a target ligand in place of the growthfactor (IL-3, in this case) free medium described above.

Example 6 Construction of an Expression System of Secretary and SolubleRecombinant NR12 Protein

Though rare, cell membrane-binding proteins except soluble proteins canbe envisaged as a ligand specifically binding to the protein of theinvention. In such cases, screening can be done by labeling the proteincontaining only the extracellular domain of the protein of theinvention, or a fusion protein in which a partial sequence of anothersoluble protein is added to the extracellular domain of the presentprotein, and then, measuring the binding with cells expected to expressthe ligand.

Examples of the former proteins containing only the extracellular domainof the protein of the invention are, for example, soluble receptorproteins artificially prepared by inserting a stop codon to theN-terminal side of the transmembrane domain, or NR12.1 that encodes thesoluble type protein of NR12. On the other hand, the latter proteins maybe prepared by adding labeling peptide sequences such as Fc site ofimmunoglobulins, and FLAG peptide to the C-terminus of the extracellulardomain of the protein of the invention. These soluble labeled proteinscan be also used for the detection in the West-western blotting method.

The present inventors selected a construction method as follows: (1)cDNA sequence encoding the extracellular region of NR12 (amino acidsequence from the 1^(st) Met to the 319^(th) Gly) was amplified by PCR;and (2) FLAG peptide sequence was added in frame to the C-terminus ofthe amplified DNA fragment to obtain a sequence encoding the solubletargeted protein. The constructed sequence was inserted into the plasmidvector, pCHO, which can be expressed in mammalian cells. A schematicdiagram of the structure of the constructed pCHO/NR12-TPOR chimericreceptor is shown in FIG. 10. This expression vector was introduced intomammalian cells, CHO cells, and was forced to express. Then, stablegene-introduced cells were selected. After confirming expression of thesoluble protein, the expression cells were cultured in large scale. Therecombinant protein secreted into the culture supernatant can beimmunoprecipitated using anti-FLAG peptide antibody, and may be purifiedby affinity columns, etc.

The obtained recombinant protein can be applied not only for the assaymentioned above, but also, for example, for detection of specific bidingactivity within a material expected to contain a target ligand byBIA-CORE system (Pharmacia). Thus, it is extremely important forsearching novel hemopoietins that can bind to NR12.

Example 7 Reisolation of Human Full-Length NR12 CDS (1) Design ofOligonucleotide Primers

The present inventors already had succeeded in isolating the full-lengthcDNA of NR12 gene. However, the N-terminal sequence and C-terminalsequence of the isolated target gene were isolated separately due to theuse of 5′-RACE and 3′-RACE method for the cDNA isolation. Thus, thepresent inventors attempted to reisolate NR12.2 and NR12.3 genes thatcontain continuous full-length coding sequences.

First, a sense primer (NR12.2-MET) described below that contains thestart codon, Met sequence, with a common nucleotide sequence to eachcDNA clone of NR12 was designed. As the antisense primers, NR12.2-STPand NR12.3-STP that contain a stop codon specific to NR12.2 and NR12.3,respectively, were designed. The primers were synthesis as in Example 1(3). More specifically, ABI's 394 DNA/RNA Synthesizer was used for theprimer synthesis under the condition where a trityl group is attached tothe 5′-terminus. Then, the product was purified using and OPC column(ABI#400771) to obtain full-length primers.

(SEQ ID NO:18) NR12.1-MET; 5′- ATG AAT CAG GTC ACT ATT CAA TGG -3′ (SEQID NO:19) NR12.2-STP; 5′- GCA GTC CTC CTA CTT CAG CTT CCC -3′ (SEQ IDNO:20) NR12.3-STP; 5′- TTG ATT TTG ACC ACA CAG CTC TAC -3′

(2) PCR Cloning

In order to isolate the full-length CDS of NR12, PCR cloning wasperformed using NR12.1-MET primer of (1) as sense primer and NR12.2-STPand NR12.3-STP primer as antisense primers, respectively. Human ThymusMarathon-Ready cDNA Library (Clontech#7415-1) was used as the template,and Advantage cDNA Polymerase Mix (Clontech#8417-1) for the PCRexperiment on a thermal cycler (Perkin Elmer Gene Amp PCR System 2400)under the condition described below. The PCR product of 1301 bp namedNR12.4 was obtained using the primer set “NR12.1-MET and NR12.2-STP”,and that of 1910 bp named NR12.5 was obtained using the primer set“NR12.1-MET and NR12.3-STP”.

PCR was performed by a single cycle of “94° C. for 4 min”, 5 cycles of“94° C. for 20 sec, 72° C. for 90 sec”, 5 cycles of “94° C. for 20 sec,70° C. for 90 sec”, 28 cycles of “94° C. for 20 sec, 68° C. for 90 sec”,a single cycle of “72° C. for 3 min”, and was terminated at 4° C.

The obtained PCR products were subcloned into pGEM-T Easy vectors(Promega #A1360) as in Example 1 (4), and the nucleotide sequences weredetermined. Recombination of the PCR products into the pGEM-T Easyvectors were performed using T4 DNA ligase (Promega #1360) in a reactionat 4° C. for 12 hours. The recombinant of the PCR product and the pGEM-TEasy vector was obtained by transformation of E. coli strain DH5α(Toyobo #DNA-903), and Insert Check Ready Blue (Toyobo #PIK-201) wasused for the selection of the genetic recombinant. The nucleotidesequence was determined using the BigDye Terminator Cycle Sequencing SFReady Reaction Kit (ABI/Perkin Elmer #4303150) and was analyzed by theABI PRISM 377 DNA Sequencer. The nucleotide sequence of the insertfragments in respective recombinants of NR12.4 and NR12.5 were analyzed,and the sequences of cDNA clones that may encode the full-length CDSwere determined.

As a result, it was revealed that NR12.4 contains the full-length ORF ofNR12.2 but not the 5′-untranslated region or 3′-untranslated regionexcept the sequence derived from primers due to the design of the usedprimers in the PCR. NR12.5 also contained the full-length ORF of NR12.3but not the 5′-untranslated region or 3′-untranslated region except thesequence derived from the primers. The determined nucleotide sequence ofNR12.4 and its amino acid sequence are shown in FIGS. 11 and 12, and thedetermined nucleotide sequence of NR12.5 and its amino acid sequence areshown in FIGS. 13 and 14.

E. coli strain DH5α transfected with pGEM-T Easy vector (pGEM/NR12.5CDS)that contains the NR12.5 cDNA of this invention was depositedinternationally on 31 Jul. 2000 as follows.

Name and Address of the depositary institution

Depositary institution: National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Ministryof International Trade and Industry.

Address: 1-1-3 Higashi, Tsukuba, Ibaraki 305-8566, Japan.

Deposition date: 31 Jul. 2000

Accession No.: FERM BP-7259

Example 8 Cloning of Mouse NR12 Homologous Genomic Gene (1) Preparationof a Probe Fragment of Human NR12

Aiming to analyze the genomic structure of mouse NR12 gene, the presentinventors performed plaque hybridization against the mouse genomic DNAlibrary. To perform heterologous cross hybridization cloning againstmouse genomic DNA library, probe fragment of human NR12 cDNA wasprepared. The insert fragment cut out with Not I from the 5′-RACEproduct of human NR12 obtained in Example 1 (4) was purified, and usedas the probe fragment. QIAquick Gel Extraction Kit (QIAGEN #28704) wasused to extract and purify the insert fragment from the agarose gel. Theprobe was radiolabeled with [α-³²P] dCTP using Mega Prime Kit as inExample 3, and was used for plaque hybridization.

(2) Plaque Hybridization

Mouse 129SVJ strain Genomic DNA (Stratagene #946313) constructed inLambda FIX II was used as the library. A genomic library ofapproximately 320 thousand plagues was developed in NZY agar medium, andthe plaques were blotted onto a Hybond N (+) (Amersham #RPN303B) chargednylon membrane to conduct primary screening. Perfect-Hyb Solution(Toyobo#HYB-101) was used for hybridization, and after prehybridizationat 60° C. for 30 min, heat-denatured labeled probe was added, andhybridization was conducted at 60° C. for 16 hr. After subsequent washin: (1) 1×SSC/0.1% SDS at room temperature for 5 min; (2) 1×SSC/0.1% SDSat 50° C. for 30 min; and (3) 0.5×SSC/0.1% SDS at 50° C. 30 min, themembrane was exposed to an X-ray film (Hyperfilm MP: Amersham, #RPN8H)to detect mouse NR21 positive plaques.

As a result, 6 independent positive or pseudo-positive clones wereobtained. The inventors succeeded in isolating plaques of 2 independentNR12 positive clones by performing secondary screening in a similar wayto the primary screening against these 6 clones obtained by the primaryscreening. Lambda DNA of the isolated plaque was prepared in large scaleby plate-lysing method. The insert fragments were cut out withrestriction enzyme Sal I. Analysis of their size revealed that thefragments were approximately 18.5 kb and 16.0 kb, respectively.

INDUSTRIAL APPLICABILITY

The present invention provides novel hemopoietin receptor proteins andDNA encoding same. The present invention also provides: a vector intowhich the DNA has been inserted, a transformant harboring the DNA, and amethod for producing recombinant proteins using the transformant. Itfurther provides a method of screening for a compound or a naturalligand that binds to the protein. The protein of this invention ispredicted to be associated with the regulation of immune system andhematopoiesis. Therefore, the proteins of this invention are expected tobe useful in understanding immune responses and fundamental features ofhematopoiesis in vivo. It is also expected that the proteins of thepresent invention can be used in the diagnosis and treatment of diseasesrelated to immunity and hematopoiesis.

It is important to isolate unknown hematopoietic factors that can bindto the NR12 molecule of this invention. The gene of this invention isthought to be extremely useful in the screening of such unknown factors.Furthermore, peptide libraries and synthetic chemical materials may besearched to isolate and identify agonists and antagonists that canfunctionally bind to the NR12.

As described above, the NR12 gene is expected to provide a useful sourcefor obtaining unknown hematopoietic factors or agonists that are capableof functionally binding to the receptor protein encoded by the NR12gene. It is expected that cellular immunity and hematopoietic functionin vivo will be enhanced by the administration of such functionallybinding substances or specific antibodies that can activate the functionof NR12 molecule to the organism. Thus, the NR12 gene facilitates thedevelopment of drugs for clinical application that promote proliferationor differentiation of the immune cells or hematopoietic cells, or thatactivates the function of immune cells. Such drugs may be used toenhance cytotoxic immunity against specific types of tumor. It ispossible that NR12 is expressed in a restricted population of cells inthe hematopoietic tissues. Accordingly, anti-NR12 antibodies would beuseful in the isolation of such cell populations, which may then be usedin cell transplantation treatments.

On the other hand, NR12.1, a splice variant of NR12, may be used as aninhibitor for the NR12 ligand, as a decoy type receptor. Further, it isexpected that by administering antagonists that can bind functionally tothe NR12 molecule, or other inhibitors, as well as specific antibodiesthat can inhibit the molecular function of NR12 to the organism, one canpotentially suppress cellular immunity or inhibit the proliferation ofhematopoietic cells in vivo. Thus, such inhibitors may be applied asdrugs for clinical application for use as, for example, proliferationinhibitors of immune cell and hematopoietic cell, differentiationinhibitors, immunosuppressive drugs, and anti-inflammatory drugs.Specifically, such inhibitors may be used to suppress the onset ofautoimmune diseases arising from autoimmunity, or tissue rejection bythe immune system of the living body, the primary problem intransplantation. Furthermore, the inhibitors may be effectively used totreat diseases caused by such aberrant promotion of immune response.Thus, the inhibitors may be used to treat a variety of allergies thatare specific to particular antigens, such as metal and pollen.

1. A purified antibody or antigen-binding fragment thereof that bindsspecifically to a polypeptide consisting of the amino acid sequence ofSEQ ID NO:2.
 2. A purified antibody or fragment thereof that bindsspecifically to a polypeptide consisting of the amino acid sequence fromGly at position 24 to Cys at position 337 of SEQ ID NO:2.
 3. Theantibody or fragment thereof of claim 1, wherein the antibody is amonoclonal antibody.
 4. The antibody or fragment thereof of claim 2,wherein the antibody is a monoclonal antibody.
 5. The antibody orfragment thereof of claim 1, wherein the antibody is a chimericantibody.
 6. The antibody or fragment thereof of claim 2, wherein theantibody is a chimeric antibody.
 7. The antibody or fragment thereof ofclaim 1, wherein the antibody is a humanized antibody.
 8. The antibodyor fragment thereof of claim 2, wherein the antibody is a humanizedantibody.
 9. The antibody or fragment thereof of claim 1, wherein theantibody is a human antibody.
 10. The antibody or fragment thereof ofclaim 2, wherein the antibody is a human antibody.
 11. A modifiedantibody or antigen-binding fragment thereof that binds specifically toa polypeptide consisting of the amino acid sequence of SEQ ID NO:2,wherein the antibody or fragment thereof is bound to a molecule usingchemical modification.
 12. A modified antibody or antigen-bindingfragment thereof that binds specifically to a polypeptide consisting ofthe amino acid sequence from Gly at position 24 to Cys at position 337of SEQ ID NO:2, wherein the antibody or fragment thereof is bound to amolecule using chemical modification.
 13. The antibody fragment of claim1, wherein the antibody fragment is a single chain Fv.
 14. The antibodyfragment of claim 2, wherein the antibody fragment is a single chain Fv.15. A method of forming an immune complex, the method comprising thesteps of: (a) providing the antibody or fragment thereof of claim 1, and(b) contacting the antibody or fragment thereof with a polypeptide thatbinds to the antibody.
 16. A method of forming an immune complex, themethod comprising the steps of: (a) providing the antibody or fragmentthereof of claim 2, and (b) contacting the antibody or fragment thereofwith a polypeptide that binds to the antibody.
 17. A method of formingan immune complex, the method comprising the steps of: (a) providing theantibody or fragment thereof of claim 5, and (b) contacting the antibodyor fragment thereof with a polypeptide that binds to the antibody.
 18. Amethod of forming an immune complex, the method comprising the steps of:(a) providing the antibody or fragment thereof of claim 6, and (b)contacting the antibody or fragment thereof with a polypeptide thatbinds to the antibody.
 19. The method of claim 15, further comprisingthe step of: (c) detecting the immune complex.
 20. The method of claim16, further comprising the step of: (c) detecting the immune complex.21. The method of claim 17, further comprising the step of: (c)detecting the immune complex.
 22. The method of claim 18, furthercomprising the step of: (c) detecting the immune complex.